Animal product free system and process for purifying a botulinum toxin

ABSTRACT

Chromatographic processes and systems for purifying a botulinum toxin from an APF fermentation medium.

CROSS REFERENCE OF RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/136,561, filed Dec. 20, 2013, now U.S. Pat. No. 9,725,705, which is aContinuation Application of U.S. application Ser. No. 13/781,126, filedFeb. 28, 2013, now U.S. Pat. No. 8,841,110, which is a Continuation ofU.S. application Ser. No. 12/098,896, filed Apr. 7, 2008, now U.S. Pat.No. 8,409,828, which is a Continuation of U.S. application Ser. No.11/072,673, filed Mar. 3, 2005, now U.S. Pat. No. 7,354,740, which is aContinuation in Part of U.S. patent application Ser. No. 11/072,050,filed Mar. 3, 2005, now U.S. Pat. No. 7,160,699, which is a Continuationin Part of U.S. application Ser. No. 10/672,876, filed Sep. 25, 2003,now U.S. Pat. No. 7,148,041, the entire contents of applications whichare incorporated herein by reference.

BACKGROUND

The present invention relates to systems and processes for purifying aClostridium toxin. In particular, the present invention relates to achromatographic process for purifying a botulinum neurotoxin. Apharmaceutical composition suitable for administration to a human oranimal for a therapeutic, diagnostic, research or cosmetic purpose cancomprise an active ingredient. The pharmaceutical composition can alsoinclude one or more excipients, buffers, carriers, stabilizers,preservatives and/or bulking agents. The active ingredient in apharmaceutical composition can be a biologic such as a botulinum toxin.The botulinum toxin active ingredient used to make a botulinum toxinpharmaceutical composition can be obtained through a multi stepculturing, fermentation and compounding process which makes use of oneor more animal derived products (such as meat broth and caseiningredients in one or more of the culture and fermentation media used toobtain a bulk botulinum toxin, and a blood fraction or blood derivativeexcipient in the final compounded botulinum toxin pharmaceuticalcomposition). Administration to a patient of a pharmaceuticalcomposition wherein the active ingredient biologic is obtained through aprocess which makes use of animal derived products can subject thepatient to a potential risk of receiving various pathogens or infectiousagents. For example, prions may be present in a pharmaceuticalcomposition. A prion is a proteinaceous infectious particle which ishypothesized to arise as an abnormal conformational isoform from thesame nucleic acid sequence which makes the normal protein. It has beenfurther hypothesized that infectivity resides in a “recruitmentreaction” of the normal isoform protein to the prion protein isoform ata post translational level. Apparently, the normal endogenous cellularprotein is induced to misfold into a pathogenic prion conformation.

Creutzfeldt-Jacob disease is a rare neurodegenerative disorder of humantransmissible spongiform encephalopathy where the transmissible agent isapparently an abnormal isoform of a prion protein. An individual withCreutzfeldt-Jacob disease can deteriorate from apparent perfect healthto akinetic mutism within six months. Thus, a potential risk may existof acquiring a prion mediated disease, such as Creutzfeldt-Jacobdisease, from the administration of a pharmaceutical composition whichcontains a biologic, such as a botulinum toxin, obtained, purified orcompounded using animal derived products.

Botulinum Toxin

The genus Clostridium has more than one hundred and twenty sevenspecies, grouped by morphology and function. The anaerobic, grampositive bacterium Clostridium botulinum produces a potent polypeptideneurotoxin, botulinum toxin, which causes a neuroparalytic illness inhumans and animals known as botulism. Clostridium botulinum and itsspores are commonly found in soil and the bacterium can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the most lethal natural biological agent knownto man. About 50 picograms of botulinum toxin (purified neurotoxincomplex) type A is a LD₅₀ in mice. On a molar basis, botulinum toxintype A is 1.8 billion times more lethal than diphtheria, 600 milliontimes more lethal than sodium cyanide, 30 million times more lethal thancobrotoxin and 12 million times more lethal than cholera. Singh,Critical Aspects of Bacterial Protein Toxins, pages 63-84 (chapter 4) ofNatural Toxins II, edited by B. R. Singh et al., Plenum Press, New York(1976) (where the stated LD₅₀ of botulinum toxin type A of 0.3 ng equals1 U is corrected for the fact that about 0.05 ng of BOTOX® equals 1unit). BOTOX® is the trademark of a botulinum toxin type A purifiedneurotoxin complex available commercially from Allergan. Inc., ofIrvine, Calif. One unit (U) of botulinum toxin is defined as the LD₅₀upon intraperitoneal injection into female Swiss Webster mice weighingabout 18-20 grams each. In other words, one unit of botulinum toxin isthe amount of botulinum toxin that kills 50% of a group of female SwissWebster mice. Seven generally immunologically distinct botulinumneurotoxins have been characterized, these being respectively botulinumneurotoxin serotypes A, B. C₁, D, E, F and Geach of which isdistinguished by neutralization with type-specific antibodies. Thedifferent serotypes of botulinum toxin vary in the animal species thatthey affect and in the severity and duration of the paralysis theyevoke. For example, it has been determined that botulinum toxin type Ais 500 times more potent, as measured by the rate of paralysis producedin the rat, than is botulinum toxin type B. Additionally, botulinumtoxin type B has been determined to be non-toxic in primates at a doseof 480 U/kg which is about 12 times the primate LD₅₀ for botulinum toxintype A. The botulinum toxins apparently bind with high affinity tocholinergic motor neurons, are translocated into the neuron and blockthe presynaptic release of acetylcholine.

Botulinum toxins have been used in clinical settings for the treatmentof e.g. neuromuscular disorders characterized by hyperactive skeletalmuscles. Botulinum toxin type A has been approved by the U.S. Food andDrug Administration for the treatment of essential blepharospasm,strabismus and hemifacial spasm in patients over the age of twelve, forthe treatment of cervical dystonia and for the treatment of glabellarline (facial) wrinkles. The FDA has also approved a botulinum toxin typeB for the treatment of cervical dystonia. Clinical effects of peripheralinjection (i.e. intramuscular or subcutaneous) botulinum toxin type Aare usually seen within one week of injection, and often within a fewhours after injection. The typical duration of symptomatic relief (i.e.flaccid muscle paralysis) from a single intramuscular injection ofbotulinum toxin type A can be about three months to about six months.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. Botulinum toxin A is a zincendopeptidase which can specifically hydrolyze a peptide linkage of theintracellular, vesicle associated protein SNAP-25. Botulinum type E alsocleaves the 25 kiloDalton (kD) synaptosomal associated protein(SNAP-25), but targets different amino acid sequences within thisprotein, as compared to botulinum toxin type A. Botulinum toxin types B,D, F and G act on vesicle-associated protein (VAMP, also calledsynaptobrevin), with each serotype cleaving the protein at a differentsite. Finally, botulinum toxin type C₁ has been shown to cleave bothsyntaxin and SNAP-25. These differences in mechanism of action mayaffect the relative potency and/or duration of action of the variousbotulinum toxin serotypes.

Regardless of serotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least three steps or stages. Inthe first step of the process, the toxin binds to the presynapticmembrane of the target neuron through a specific interaction between theheavy chain (H chain) and a cell surface receptor; the receptor isthought to be different for each serotype of botulinum toxin. Thecarboxyl end segment of the H chain, H_(C), appears to be important fortargeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This last step is thought to be mediated by the amino end segmentof the H chain, H_(N), which triggers a conformational change of thetoxin in response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxinthen translocates through the endosomal membrane into the cytosol.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the H and L chain. Theentire toxic activity of botulinum and botulinum toxins is contained inthe L chain of the holotoxin; the L chain is a zinc (Zn++) endopeptidasewhich selectively cleaves proteins essential for recognition and dockingof neurotransmitter-containing vesicles with the cytoplasmic surface ofthe plasma membrane, and fusion of the vesicles with the plasmamembrane. Botulinum neurotoxin, botulinum toxin B, D, F, and G causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the VAMPpresent at the cytosolic surface of the synaptic vesicle is removed as aresult of any one of these cleavage events. Each toxin specificallycleaves a different bond.

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kDInterestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with one or more associated non-toxin proteins. Thus, thebotulinum toxin type A complex can be produced by Clostridial bacteriumas 900 kD, 500 kD and 300 kD forms (approximate molecular weights).Botulinum toxin types B and C₁ are apparently produced as only a 500 kDcomplex. Botulinum toxin type D is produced as both 300 kD and 500 kDcomplexes. Finally, botulinum toxin types E and F are produced as onlyapproximately 300 kD complexes. The complexes (i.e. molecular weightgreater than about 150 kD) are believed to contain a non-toxinhemagglutinin protein and a non-toxin and non-toxic nonhemagglutininprotein. Thus, a botulinum toxin complex can comprise a botulinum toxinmolecule (the neurotoxic component) and one or more non toxic,hemagluttinin proteins and/or non toxin, non hemagluttinin proteins (thelater can be referred to as NTNH proteins) These two types of non-toxinproteins (which along with the botulinum toxin molecule can comprise therelevant neurotoxin complex) may act to provide stability againstdenaturation to the botulinum toxin molecule and protection againstdigestive acids when toxin is ingested. Additionally, it is possiblethat the larger (greater than about 150 kD molecular weight) botulinumtoxin complexes may result in a slower rate of diffusion of thebotulinum toxin away from a site of intramuscular injection of abotulinum toxin complex. The toxin complexes can be dissociated intotoxin protein and hemagglutinin proteins by treating the complex withred blood cells at pH 7.3, or by subjecting the complex to a separationprocess, such as column chromatography, in a suitable buffer at a pH ofabout 7-8. The botulinum toxin protein has a marked instability uponremoval of the hemagglutinin protein.

All the botulinum toxin serotypes are made by native Clostridiumbotulinum bacteria as inactive single chain proteins which must becleaved or nicked by proteases to become neuroactive. The bacterialstrains that make botulinum toxin serotypes A and G possess endogenousproteases and serotypes A and G can therefore be recovered frombacterial cultures in predominantly their active form. In contrast,botulinum toxin serotypes C₁, D, and E are synthesized by nonproteolyticstrains and are therefore typically unactivated when recovered fromculture. Serotypes B and F are produced by both proteolytic andnonproteolytic strains and therefore can be recovered in either theactive or inactive form. However, even the proteolytic strains thatproduce, for example, the botulinum toxin type B serotype only cleave aportion of the toxin produced. The exact proportion of nicked tounnicked molecules depends on the length of incubation and thetemperature of the culture. Therefore, a certain percentage of anypreparation of, for example, the botulinum toxin type B toxin is likelyto be inactive, possibly accounting for the known significantly lowerpotency of botulinum toxin type B as compared to botulinum toxin type A.The presence of inactive botulinum toxin molecules in a clinicalpreparation will contribute to the overall protein load of thepreparation, which has been linked to increased antigenicity, withoutcontributing to its clinical efficacy. Additionally, it is known thatbotulinum toxin type B has, upon intramuscular injection, a shorterduration of activity and is also less potent than botulinum toxin type Aat the same dose level.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes. Thus,at least botulinum toxins types, A, B. E and F have been used clinicallyin humans. Additionally, pure (approx 150 kDa) botulinum toxin has beenused to treat humans. See e.g. Kohl A., et al, Comparison of the effectof botulinum toxin A (Botox®) with the highly-purified neurotoxin (NT201) in the extensor digitorum brevis muscle test. Mov Disord 2000;15(Suppl 3):165. Hence, a botulinum toxin pharmaceutical composition canbe prepared using a pure (approx 150 kDa) botulinum toxin, as opposed touse of a botulinum toxin complex.

The type A botulinum toxin is known to be soluble in dilute aqueoussolutions at pH 4-6.8. At pH above about 7 the stabilizing nontoxicproteins dissociate from the neurotoxin, resulting in a gradual loss oftoxicity, particularly as the pH and temperature rise. Schantz E. J., etal Preparation and characterization of botulinum toxin type A for humantreatment (in particular pages 44-45), being chapter 3 of Jankovic, J.,et al, Therapy with Botulinum Toxin, Marcel Dekker, Inc (1994).

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) is dependant, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of the toxincomplex obtained by the known culturing, fermentation and purificationto the much, much lower toxin concentrations used for pharmaceuticalcomposition formulation results in rapid detoxification of the toxinunless a suitable stabilizing agent is present. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties because of the rapid lossof specific toxicity upon such great dilution. Since the toxin may beused months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin can be stabilized with astabilizing agent such as albumin and gelatin.

It has been reported that a botulinum toxin has been used in variousclinical settings, including as follows:

(1) about 75-125 units of BOTOX® per intramuscular injection (multiplemuscles) to treat cervical dystonia;(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercilii muscle);(3) about 30-80 units of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle;(4) about 1-5 units per muscle of intramuscularly injected BOTOX® totreat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:

(a) flexor digitorum profundus: 7.5 U to 30 U

(b) flexor digitorum sublimus: 7.5 U to 30 U

(c) flexor carpi ulnaris: 10 U to 40 U

(d) flexor carpi radialis: 15 U to 60 U

(e) biceps brachii: 50 U to 200 U. Each of the five indicated muscleshas been injected at the same treatment session, so that the patientreceives from 90 U to 360 U of upper limb flexor muscle BOTOX® byintramuscular injection at each treatment session.

(7) to treat migraine, pericranial injected (injected symmetrically intoglabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX®has showed significant benefit as a prophylactic treatment of migrainecompared to vehicle as measured by decreased measures of migrainefrequency, maximal severity, associated vomiting and acute medicationuse over the three month period following the 25 U injection.

It is known that botulinum toxin type A can have an efficacy for up to12 months (European J. Neurology 6 (Supp 4): S111-S1150:1999), and insome circumstances for as long as 27 months. The Laryngoscope109:1344-1346:1999. However, the usual duration of an intramuscularinjection of Botox® is typically about 3 to 4 months.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, human serum albumin, and sodium chloride packaged insterile, vacuum-dried form. The botulinum toxin type A is made from aculture of the Hall strain of Clostridium botulinum grown in a mediumcontaining N-Z amine casein and yeast extract. The botulinum toxin typeA complex is purified from the culture solution by a series of acid oracid and ethanol precipitations to a crystalline complex consisting ofthe active high molecular weight toxin protein and an associatedhemagglutinin protein. The crystalline complex is re-dissolved in asolution containing saline and albumin and sterile filtered (0.2microns) prior to vacuum-drying. BOTOX® can be reconstituted withsterile, non-preserved saline prior to intramuscular injection. Eachvial of BOTOX® contains about 100 units (U) of Clostridium botulinumtoxin type A complex, 0.5 milligrams of human serum albumin and 0.9milligrams of sodium chloride in a sterile, vacuum-dried form without apreservative.

To reconstitute vacuum-dried BOTOX® sterile normal saline without apreservative (0.9% Sodium Chloride injection) is used by drawing up theproper amount of diluent in the appropriate size syringe. Since BOTOX®is denatured by bubbling or similar violent agitation, the diluent isgently injected into the vial. Reconstituted BOTOX® can be stored in arefrigerator (2° to 8° C.) and is a clear, colorless liquid and free ofparticulate matter. There are reports of reconstituted BOTOX® retainingits potency for up to thirty days. See e.g. Guttman C., Botox retainsits efficacy for blepharospasm treatment after freezing and storage, NewYork investigators find, EuroTimes 2000 November/December; 5(8):16. Thevacuum-dried product is stored in a freezer at or below −5° C.

In general, four physiologic groups of C. botulinum are recognized (I,II, III, IV). The organisms capable of producing a serologicallydistinct toxin may come from more than one physiological group. Forexample, Type B and F toxins can be produced by strains from Group I orII. In addition, other strains of clostridial species (C. baratii, typeF; C. butyricum, type E; C. novyi, type C₁ or D) have been identifiedwhich can produce botulinum neurotoxins.

The physiologic groups of Clostridium botulinum types are listed inTable 1.

Physiologic Groups of Closrtidium botulinum Pheno- Glu- typically coseRelated Tox- Fer- Phages Clos- in Milk men- & tridium Sero- Bio Di- ta-Li- Plas- (non- Group Type chemistry gest tion pase mids toxigenic) I A,proteolytic + + + + C. B, F saccharolytic sporogenes II B, non- − + + +E, F proteolytic saccharolytic psycho- trophic III C,D Non- ± + + + C.novyi proteolytic saccharolytic IV G proteolytic + − − − C. non-subterminale saccharolytic

These toxin types may be produced by selection from the appropriatephysiologic group of Clostridium botulinum organisms. The organismsdesignated as Group I are usually referred to as proteolytic and producebotulinum toxins of types A, B and F. The organisms designated as GroupII are saccharolytic and produce botulinum toxins of types B, E and F.The organisms designated as Group III produce only botulinum toxin typesC and D and are distinguished from organisms of Groups I and II by theproduction of significant amounts of propionic acid. Group IV organismsproduce only neurotoxin of type G.

It is known to obtain a tetanus toxin using specific media substantiallyfree of animal products. See e.g. U.S. Pat. No. 6,558,926. But notably,even the “animal product free” media disclosed by this patent usesBacto-peptone, a meat digest. Significantly, production of tetanus toxinby Clostridium tetani vs. production of a botulinum toxin by aClostridium botulinum bacterium entails different growth, media andfermentation parameters and considerations. See e.g. Johnson, E. A., etal, Clostridium botulinum and its neurotoxins: a metabolic and cellularperspective, Toxicon 39 (2001), 1703-1722.

Production of Active Botulinum Neurotoxin

Botulinum toxin for use in a pharmaceutical composition can be obtainedby anaerobic fermentation of Clostridium botulinum using a modifiedversion of the well known Schantz process (see e.g. Schantz E. J., etal., Properties and use of botulinum toxin and other microbialneurotoxins in medicine, Microbiol Rev 1992 March; 56(1):80-99; SchantzE. J., et al., Preparation and characterization of botulinum toxin typeA for human treatment, chapter 3 in Jankovic J, ed. Neurological Diseaseand Therapy, Therapy with botulinum toxin (1994), New York, MarcelDekker 1994, pages 41-49, and; Schantz E. J., et al., Use of crystallinetype A botulinum toxin in medical research, in: Lewis G E Jr. ed.Biomedical Aspects of Botulism (1981) New York, Academic Press, pages143-50.).

A Clostridium botulinum neurotoxin (as pure toxin or as a botulinumtoxin complex) can also be obtained by aerobic fermentation of arecombinant host cell which bears the appropriate gene. See e.g. U.S.Pat. No. 5,919,665 entitled Vaccine for Clostridium botulinumneurotoxin, issued Jul. 6, 1999 to Williams and U.S. patent application20030215468 entitled Soluble recombinant botulinum toxin proteins byWilliams et al., published Nov. 20, 2003.

Additionally, botulinum toxins (the 150 kilodalton molecule) andbotulinum toxin complexes (300 kDa to 900 kDa) can be obtained from, forexample. List Biological Laboratories, Inc., Campbell, Calif.; theCentre for Applied Microbiology and Research, Porton Down, U.K., Wako(Osaka, Japan), as well as from Sigma Chemicals of St Louis, Mo.Commercially available botulinum toxin containing pharmaceuticalcompositions include Botox® (Botulinum toxin type A purified neurotoxincomplex with human serum albumin and sodium chloride) available fromAllergan, Inc., of Irvine, Calif. in 100 unit vials as a lyophilizedpowder to be reconstituted with 0.9% sodium chloride before use),Dysport® (Clostridium botulinum type A toxin hemagglutinin complex withhuman serum albumin and lactose in the botulinum toxin pharmaceuticalcomposition), available from Ipsen Limited, Berkshire, U.K. as a powderto be reconstituted with 0.9% sodium chloride before use), and MyoBloc™(an injectable solution comprising botulinum toxin type B, human serumalbumin, sodium succinate, and sodium chloride at about pH 5.6,available from Solstice Neurosciences (formerly available from ElanCorporation, Dublin, Ireland) of San Diego, Calif.

A number of steps are required to make a Clostridial toxinpharmaceutical composition suitable for administration to a human oranimal for a therapeutic, diagnostic, research or cosmetic purpose.These steps can include obtaining a purified Clostridial toxin and thencompounding the purified Clostridial toxin. A first step can be toculture a Clostridial bacteria, typically on agar plates, in anenvironment conducive to bacterial growth, such as in a warm anaerobicatmosphere. The culture step allows Clostridial colonies with desirablemorphology and other characteristics to be obtained. In a second stepselected cultured Clostridial colonies can be fermented in a suitablemedium. After a certain period of fermentation the Clostridial bacteriatypically lyse and release Clostridial toxin into the medium. Thirdly,the culture medium can be purified so as to obtain a bulk or raw toxin.Typically culture medium purification to obtain bulk toxin is carriedout using, among other reagents, animal derived enzymes, such as DNaseand RNase, which are used to degrade and facilitate removal of nucleicacids. The resulting bulk toxin can be a highly purified toxin with ahigh specific activity. After stabilization in a suitable buffer, thebulk toxin can be compounded with one or more excipients to make aClostridial toxin pharmaceutical composition suitable for administrationto a human. The Clostridial toxin pharmaceutical composition cancomprises a Clostridial toxin as an active pharmaceutical ingredient.The pharmaceutical composition can also include one or more excipients,buffers, carriers, stabilizers, preservatives and/or bulking agents.

The Clostridium toxin fermentation step can result in a culture solutionwhich contains whole Clostridium bacteria, lysed bacteria, culture medianutrients and fermentation byproducts Filtration of this culturesolution so as to remove gross elements, such as whole and lysedbacteria, provides a clarified culture. The clarified culture solutioncomprises a Clostridial and various impurities and can be processed soas to obtain a concentrated Clostridial toxin, which is called bulktoxin.

Fermentation and purification processes for obtaining a bulk Clostridialtoxin using one or more animal derived products (such as the milk digestcasein, DNase and RNase) are known. An example of such a known non-APFprocess for obtaining a botulinum toxin complex is the Schantz process.The Schantz process (from initial cell culture through to fermentationand toxin purification) makes use of a number of products derived fromanimal sources such as for example animal derived Bacto Cooked Meatmedium in the culture vial, Columbia Blood Agar plates for colony growthand selection, and casein in the fermentation media. Additionally, theSchantz bulk toxin purification process makes use of DNase and RNasefrom bovine sources to hydrolyze nucleic acids present in the toxincontaining fermented culture medium.

A fermentation process for obtaining a tetanus toxoid which uses reducedamounts of animal derived products (referred to as animal protein freeor “APF” fermentation processes) is known. See e.g. U.S. Pat. No.6,558,926 entitled Method for production of tetanus toxin using mediasubstantially free of animal products, issued to Demain et al., May 6,2003. An APF fermentation process for obtaining a Clostridial toxin, hasthe potential advantage of reducing the (the already very low)possibility of contamination of the ensuing bulk toxin with viruses,prions or other undesirable elements which can then accompany the activepharmaceutical ingredient Clostridial toxin as it is compounded into apharmaceutical composition for administration to humans.

It is known to use chromatography to purify a Clostridial toxin. Thus:

-   1. Ozutsumi K., et al, Rapid, simplified method for production and    purification of tetanus toxin, App & Environ Micro, April 1985, p    939-943, vol 49, no. 4.(1985) discloses use of high pressure liquid    chromatography (HPLC) gel filtration to purify tetanus toxin.-   2. Schmidt J. J., et al., Purification of type E botulinum    neurotoxin by high-performance ion exchange chromatography, Anal    Biochem 1986 July:156(1):213-219 discloses use of size exclusion    chromatography or ion exchange chromatograph to purify botulinum    toxin type E. Also disclosed is use of protamine sulfate instead of    ribonuclease (RNase).-   3. Simpson L. L., et al, Isolation and characterization of the    botulinum neurotoxins Simpson L L; Schmidt J J; Middlebrook J L. In:    Harsman S, ed. Methods in Enzymology. Vol. 165, Microbial Toxins:    Tools in Enzymology San Diego, Calif.: Academic Press; vol 165:pages    76-85 (1988) discloses purification of botulinum neurotoxins using    gravity flow chromatography, HPLC, capture steps using an affinity    resin, size exclusion chromatography, and ion (anion and cation)    exchange chromatography, including use of two different ion exchange    columns Various Sephadex, Sephacel. Trisacryl, S and Q columns are    disclosed.-   4. Zhou L, et al., Expression and purification of the light chain of    botulinum neurotoxin A: A single mutation abolishes its cleavage of    SNAP-25 and neurotoxicity after reconstitution with the heavy chain,    Biochemistry 1995; 34(46):15175-81 (1995) discloses use of an    amylose affinity column to purify botulinum neurotoxin light chain    fusion proteins.-   5. Kannan K., et al., Methods development for the biochemical    assessment of Neurobloc (botulinum toxin type B), Mov Disord 2000;    15(Suppl 2):20 (2000) discloses use of size exclusion chromatography    to assay a botulinum toxin type B.-   6. Wang Y-c, The preparation and quality of botulinum toxin type A    for injection (BTXA) and its clinical use, Dermatol Las Faci Cosm    Surg 2002; 58 (2002) discloses ion exchange chromatography to purify    a botulinum toxin type A. This reference discloses a combination of    precipitation and chromatography techniques.-   7. Johnson S. K., et al., Scale-up of the fermentation and    purification of the recombination heavy chain fragment C of    botulinum neurotoxin serotype F, expressed in Pichia pastoris,    Protein Expr and Purif 2003:32:1-9 (2003) discloses use of ion    exchange and hydrophobic interaction columns to purify a recombinant    heavy chain fragment of a botulinum toxin type F.-   8. Published U.S. patent application 2003 0008367 A1 (Oguma)    discloses use of ion exchange and lactose columns to purify a    botulinum toxin.

The purification methods summarized above relate generally to researchor laboratory scale methods which are not scaleable into industrial orcommercial processes. It is well known that chromatography techniquessuch as, for example, gel filtration and gravity flow chromatography arenot amenable for use as large-scale, validatable, cGMP manufacturingprocesses. Alternately or in addition, the purification methodsummarized above relate to small scale purification of the pure toxin(i.e. the approximately 150 kDa neurotoxic molecule), or a specificcomponent of the neurotoxic, as opposed of the entire 900 kDa botulinumtoxin complex. As is also well known, obtaining a biologically active,purified botulinum toxin complex is considerably more complex anddifficult, than is purifying only a component of the complex. This isdue, for example, to the larger size, fragility, labile nature andparticular secondary, tertiary and quaternary molecule and complexconformations required for obtaining a biologically active and stablebotulinum toxin complex.

Furthermore, existing processes, including commercial scale processes,for obtaining a botulinum toxin suitable for compounding into abotulinum toxin pharmaceutical composition typically include a series ofprecipitation steps to separate the toxin complex from impurities whichaccompany the botulinum toxin from the fermentation process. Notably,precipitation techniques are widely used in the biopharmaceuticalindustry to purify a botulinum toxin. For example, cold alcoholfractionation (Cohn's method) or precipitation is used to remove plasmaproteins. Unfortunately, precipitation techniques for purifying abotulinum toxin have the drawbacks of low resolution, low productivity,difficulty to operate, difficulty to control and/or validate, anddifficulty to scale-up or scale-down.

What is needed therefore is an APF process for purifying a Clostridialtoxin fermentation medium so as to obtain a bulk Clostridial toxinwithout making use of animal derived products in the purificationprocess.

SUMMARY

Our invention provides various chromatographic APF systems and processesfor purifying a Clostridial toxin. The systems and processes of ourinvention are scalable and cGMP compliant. The Clostridial toxin ispreferably a botulinum toxin, and most preferably a botulinum toxin typeA 900 kDa complex. The present invention can be used as a commercial,industrial scale APF purification process, to purify the Clostridialtoxin (such as botulinum toxin) obtained from a separate APFfermentation (i.e. use of soy instead of casein in the fermentationmedium) of a Clostridial bacterium. The present invention thereforepermits replacement of the non-APF purification (i.e. use of DNase andRNase) process, which is typically carried out after a non-APFfermentation, to purify the botulinum toxin.

The present invention can also have utility subsequent to a Schantzfermentation of a Clostridial bacterium, to replace the Schantz(non-APF) purification process, with the herein disclosed APF toxinpurification process. It is not preferred to practice the presentinvention after a non-APF fermentation process, as opposed to practicingthe present invention after an APF fermentation process, because thepresent invention has been optimized for use subsequent to an APFfermentation process.

Thus, processes within the scope of the present invention are preferablyused in conjunction with (subsequent to) an APF fermentation to therebyfurther reduce, and in certain embodiments eliminate, use of animalderived products in the steps required to obtain a bulk Clostridialtoxin. Clearly practice of the present invention subsequent to an APFfermentation process permits an essentially completely APF methodology(fermentation and purification) to be carried out.

An embodiment of the present invention provides a system and process forobtaining high yield of highly purified biologically active Clostridialtoxin. The present invention accomplishes this through use of a free orsubstantially animal product free chromatographic system and process topurify a clarified culture obtained from the fermentation processes of aClostridium bacterium, such as a Clostridium botulinum bacterium.

Definitions

As used herein, the words or terms set forth below have the followingdefinitions.

“About” means that the item, parameter or term so qualified encompassesa range of plus or minus ten percent above and below the value of thestated item, parameter or term.

“Administration,” or “to administer” means the step of giving (i.e.administering) a pharmaceutical composition to a subject. Thepharmaceutical compositions disclosed herein are “locally administered”by e.g. intramuscular (i.m.), intradermal, subcutaneous administration,intrathecal administration, intracranial, intraperitoneal (i.p.)administration, topical (transdermal) and implantation (i.e. of aslow-release device such as polymeric implant or miniosmotic pump)routes of administration.

“Animal product free” or “substantially animal product free”encompasses, respectively, “animal protein free” or “substantiallyanimal protein free” and means the absence or substantial absence ofblood derived, blood pooled and other animal derived products orcompounds. “Animal” means a mammal (such as a human), bird, reptile,fish, insect, spider or other animal species. “Animal” excludesmicroorganisms, such as bacteria. Thus, an animal product free medium orprocess or a substantially animal product free medium or process withinthe scope of the present invention can include a botulinum toxin or aClostridial botulinum bacterium. For example, an animal product freeprocess or a substantially animal product free process means a processwhich is either substantially free or essentially free or entirely freeof animal derived proteins, such as immunoglobulins, meat digest, meatby products and milk or dairy products or digests. Thus, an example ofan animal product free process is a process (such as a bacterialculturing or bacterial fermentation process) which excludes meat anddairy products or meat or dairy by products.

“Botulinum toxin” means a neurotoxin produced by Clostridium botulinum,as well as modified, recombinant, hybrid and chimeric botulinum toxins.A recombinant botulinum toxin can have the light chain and/or the heavychain thereof made recombinantly by a non-Clostridial species.“Botulinum toxin,” as used herein, encompasses the botulinum toxinserotypes A, B, C, D, E, F and G. “Botulinum toxin,” as used herein,also encompasses both a botulinum toxin complex (i.e. the 300, 600 and900 kDa complexes) as well as pure botulinum toxin (i.e. the about 150kDa neurotoxic molecule), all of which are useful in the practice of thepresent invention. “Purified botulinum toxin” means a pure botulinumtoxin or a botulinum toxin complex that is isolated, or substantiallyisolated, from other proteins and impurities which can accompany thebotulinum toxin as it is obtained from a culture or fermentationprocess. Thus, a purified botulinum toxin can have at least 90%,preferably more than 95%, and most preferably more than 99% of thenon-botulinum toxin proteins and impurities removed. The botulinum C₂and C₃ cytotoxins, not being neurotoxins, are excluded from the scope ofthe present invention.

“Clostridial neurotoxin” means a neurotoxin produced from, or native to,a Clostridial bacterium, such as Clostridium botulinum, Clostridiumbutyricum or Clostridium beratti, as well as a Clostridial neurotoxinmade recombinantly by a non-Clostridial species.

“Entirely free (i.e. “consisting of” terminology) means that within thedetection range of the instrument or process being used, the substancecannot be detected or its presence cannot be confirmed.

“Essentially free” (or “consisting essentially of”) means that onlytrace amounts of the substance can be detected.

“Modified botulinum toxin” means a botulinum toxin that has had at leastone of its amino acids deleted, modified, or replaced, as compared to anative botulinum toxin. Additionally, the modified botulinum toxin canbe a recombinantly produced neurotoxin, or a derivative or fragment of arecombinantly made neurotoxin. A modified botulinum toxin retains atleast one biological activity of the native botulinum toxin, such as,the ability to bind to a botulinum toxin receptor, or the ability toinhibit neurotransmitter release from a neuron. One example of amodified botulinum toxin is a botulinum toxin that has a light chainfrom one botulinum toxin serotype (such as serotype A), and a heavychain from a different botulinum toxin serotype (such as serotype B).Another example of a modified botulinum toxin is a botulinum toxincoupled to a neurotransmitter, such as substance P.

“Patient” means a human or non-human subject receiving medical orveterinary care. Accordingly, as disclosed herein, the compositions maybe used in treating any animal, such as mammals.

“Pharmaceutical composition” means a formulation in which an activeingredient can be a botulinum toxin. The word “formulation” means thatthere is at least one additional ingredient (such as an albumin and/orsodium chloride) in the pharmaceutical composition besides a neurotoxinactive ingredient. A pharmaceutical composition is therefore aformulation which is suitable for diagnostic, therapeutic or cosmeticadministration (i.e. by intramuscular or subcutaneous injection or byinsertion of a depot or implant) to a subject, such as a human patient.The pharmaceutical composition can be: in a lyophilized or vacuum driedcondition: a solution formed after reconstitution of the lyophilized orvacuum dried pharmaceutical composition with saline or water, or: as asolution which does not require reconstitution. The active ingredientcan be one of the botulinum toxin serotypes A, B, C₁, D, E, F or G or abotulinum toxin, all of which can be made natively by Clostridialbacteria. As stated, a pharmaceutical composition can be liquid orsolid, for example vacuum-dried. The constituent ingredients of apharmaceutical composition can be included in a single composition (thatis all the constituent ingredients, except for any requiredreconstitution fluid, are present at the time of initial compounding ofthe pharmaceutical composition) or as a two-component system, forexample a vacuum-dried composition reconstituted with a diluent such assaline which diluent contains an ingredient not present in the initialcompounding of the pharmaceutical composition. A two-component systemprovides the benefit of allowing incorporation of ingredients which arenot sufficiently compatible for long-term shelf storage with the firstcomponent of the two component system. For example, the reconstitutionvehicle or diluent may include a preservative which provides sufficientprotection against microbial growth for the use period, for exampleone-week of refrigerated storage, but is not present during the two-yearfreezer storage period during which time it might degrade the toxin.Other ingredients, which may not be compatible with a Clostridial toxinor other ingredients for long periods of time, may be incorporated inthis manner; that is, added in a second vehicle (i.e. in thereconstitution fluid) at the approximate time of use. Methods forformulating a botulinum toxin active ingredient pharmaceuticalcomposition are disclosed in U.S. patent publication 2003 0118598 A1.

“Substantially free” means present at a level of less than one percentby weight of the pharmaceutical composition.

“Therapeutic formulation” means a formulation can be used to treat andthereby alleviate a disorder or a disease, such as a disorder or adisease characterized by hyperactivity (i.e. spasticity) of a peripheralmuscle.

The following abbreviations are used herein:

-   -   3:1:1 culture a botulinum toxin culture/fermentation medium        containing 3% HySoy, 1% HyYeast, and 1% glucose HySoy (Quest        product no. 5X59022) is a source of peptides made by enzymatic        hydrolysis of soy. HyYeast (HyYest, Quest product no. 5Z10102 or        5Z10313 is a baker's yeast extract.    -   5:1:1 culture a botulinum toxin culture/fermentation medium        containing 5% HySoy, 1% HyYeast, and 1% glucose.    -   API active pharmaceutical ingredient    -   APF animal product free    -   BCA bicinchoninic acid    -   CV column volume    -   DF diafiltration    -   ELISA enzyme linked immunosorbent assay. “Hc” in “Hc-ELISA means        a botulinum toxin heavy chain.    -   MLD50 the amount of a botulinum toxin which is a lethal so dose        to 50% of 18-23 gram Swiss-Weber mice injected intraperitoneally    -   SDS-PAGE sodium dodecylsulfate-polyacrylamide gel        electrophoresis    -   SEC-HPLC size exclusion high performance liquid chromatography    -   UF ultrafiltration    -   UV ultraviolet

Our invention includes a process for purifying a Clostridium toxin. Theprocess can have the four steps of obtaining a sample of a botulinumtoxin fermentation culture; contacting a first chromatography columnresin with the culture sample so as to permit capture of a botulinumtoxin by the first column; eluting the botulinum toxin from the firstcolumn, and; loading a second column chromatography column resin withthe eluent from the first chromatography column, thereby obtaining apurified botulinum toxin. By “botulinum toxin fermentation culture” itis meant a fermentation medium in which a Clostridium botulinumbacterium has been fermented so that the bacterium has releasedbotulinum toxin into the medium. The sample of a botulinum toxinfermentation culture (medium) is preferably a sample of a clarifiedculture of the fermentation medium.

The first chromatography column and the second chromatography column canbe different columns, and the two different columns can act to purify abotulinum toxin through different purification mechanisms. For example,the first chromatography column can be a hydrophobic interaction columnand the second chromatography column can be an ion exchange column.

A process for purifying a Clostridium toxin within the scope of ourinvention can also have the step after the contacting step and beforethe eluting step, of washing impurities off the first column.Additionally, a process for purifying a Clostridium toxin within thescope of our invention can also have the step after the loading step,the step of washing impurities off the second column. Furthermore, aprocess for purifying a Clostridium toxin within the scope of ourinvention can also have, after the step of washing impurities off thesecond column, the step of eluting the botulinum toxin from the secondcolumn.

Preferably, a process for purifying a Clostridium toxin within the scopeof our invention is an APF process, more preferably it is asubstantially animal protein free (“APF”) process, and more preferablyit is an essentially APF process for purifying a clostridial toxin, suchas a botulinum toxin complex.

The botulinum toxin fermentation culture used in a process for purifyinga Clostridium toxin within the scope of our invention preferably resultsfrom an APF process, more preferably results from a substantially APFprocess, and most preferably results from an essentially APF process.

Significantly, a process for purifying a Clostridium toxin within thescope of our invention can provide a yield of purified botulinum toxincomplex greater than about 50 mg per batch for each 10 liters of thebotulinum toxin fermentation culture.

A purified botulinum toxin complex obtained by practice of a process forpurifying a Clostridium toxin within the scope of our invention can havethe following characteristics: an appearance as an white to off-whitesuspension; a concentration of 2.0-3.6 mg of botulinum toxin complex perml of eluent; the ratio of absorbance at 260 nm to absorbance at 278 nm(A260/A278) is less than or equal to 0.6; a specific potency in MLD50unit/mg of between 2.4×10⁷ to 5.9×10⁷ MLD50 units per mg of the purifiedbotulinum toxin; an immunological identity to botulinum neurotoxin typeA complex; an SDS-PAGE characteristic that conforms to standard; anSEC-HPLC characteristic of 900 kDa toxin complex of >95% of the totalpeak, and; the process used to obtain such a purified botulinum toxincomplex is robust, scalable, validatable, and/or cGMP compliant.

An APF process for purifying a botulinum toxin complex within the scopeof our invention can have the steps of:

(a) obtaining a sample of a botulinum toxin fermentation culture,wherein the botulinum toxin fermentation culture results from asubstantially APF process.

(b) contacting a hydrophobic interaction chromatography column resinwith the culture sample so as to permit capture of a botulinum toxin bythe first column:

(c) washing impurities off the hydrophobic interaction chromatographycolumn:

(d) eluting the botulinum toxin from the hydrophobic interaction column(the eluting step can be followed by the step of diluting the eluentfrom the hydrophobic interaction chromatography column for a subsequention exchange chromatography).

(e) loading an ion exchange column chromatography column resin with theeluent (such as the diluted eluent from the hydrophobic interactionchromatography column) from the hydrophobic interaction chromatographycolumn:

(f) washing impurities off the ion exchange chromatography column, and;

(g) eluting the botulinum toxin from the ion change column, therebyobtaining a purified botulinum toxin through a process for purifying abotulinum toxin which is a substantially APF purification process.

The APF process set forth in the paragraph above can further comprise,after the step of obtaining a sample of a botulinum toxin fermentationculture and before the step of contacting a hydrophobic interactionchromatography column resin with the culture sample, the additional stepof conditioning the clarified culture for hydrophobic interactionchromatography. Additionally, the APF process set forth in the paragraphabove can further comprise, after the step of eluting the botulinumtoxin from the hydrophobic interaction column and before the step ofloading an ion exchange column chromatography column resin with theeluent from the hydrophobic interaction chromatography column, the stepof conditioning the eluent from hydrophobic interaction column for ionexchange chromatography.

A detailed embodiment of an APF process for purifying a botulinum toxin,the process can comprise the steps of:

(a) obtaining a sample of a botulinum toxin fermentation culture,wherein the botulinum toxin fermentation culture results from asubstantially APF process.

(b) conditioning the clarified culture for hydrophobic interactionchromatography;

(c) contacting a hydrophobic interaction chromatography column resinwith the culture sample so as to permit capture of a botulinum toxin bythe first column;

(d) washing impurities off the hydrophobic interaction chromatographycolumn:

(e) eluting the botulinum toxin from the hydrophobic interaction column;

(f) conditioning the eluent from hydrophobic interaction column for ionexchange chromatography:

(g) loading an ion exchange column chromatography column resin with theconditioned eluent from the hydrophobic interaction chromatographycolumn:

(h) washing impurities off the ion exchange chromatography column, and;

(i) eluting the botulinum toxin from the ion change column, therebyobtaining a purified botulinum toxin through a process for purifying abotulinum toxin which is a substantially APF purification process.

Also within the scope of our invention is a system for purifying aClostridium toxin, such as a botulinum toxin type A complex. Such asystem can comprise: a first chromatography column resin for capturing abotulinum toxin from a fermentation culture; an elution buffer foreluting the botulinum toxin from the first column; a second columnchromatography column resin for capturing a botulinum toxin from aneluent from the first chromatography column, and; a second elutionbuffer for eluting the botulinum toxin from the second chromatographycolumn.

DRAWINGS

Aspects of the invention are explained or illustrated by the followingdrawings.

FIG. 1 entitled N-Source (i e HySoy plus YE) % vs. Potency and pH” is agraph showing botulinum toxin activity as determined: (1) on the leftside Y axis as mouse lethal dose 50 (MLD 50), and; (2) on the left sideY axis as SNAP 25 activity, of various APF media at the elapsedfermentation times shown at the top of the bars, for APF medium pH asshown on the right side Y axis the pH, for APF media with the wt %amount of hydrolyzed soy concentrate and yeast extract concentrate asshown by the X axis. All FIG. 1 media also contained 1% by wt glucose.

FIG. 2 is a summary flow chart comparing a non-APF process for obtaininga botulinum toxin (the top half of FIG. 2) with an APF process, withinthe scope of the present invention, for obtaining a botulinum toxin (thebottom half of FIG. 2), through the cell bank creation, culture andfermentation steps. FIG. 2 omits the harvest and purification steps.

FIG. 3 is a chromatograph obtained from hydrophobic interactionchromatography of an APF clarified culture (a 3.1.1 culture) on a ButylSepharose Fast Flow column. The X axis in FIG. 3 represents the volumein ml of liquid (effluent) which has passed through the column. The Yaxis represents absorbance at 280 nm in mAU.

FIG. 4 is a chromatograph obtained from ionic exchange chromatography ofthe eluent from the FIG. 3 Butyl column on an SP Sepharose highperformance column. The axes in FIG. 4 are the same as they are for FIG.3.

FIG. 5A is an image of reduced SDS-PAGE of various fractions obtainedfrom operation of the Butyl column of FIG. 3. The left hand side of FIG.5A is marked vertically with descending molecular weights in thousandsof Daltons (kDa). The numbers 1 to 8 along the bottom border of FIG. 5Arepresents the lanes in which fractions were loaded.

FIG. 58 is an image of reduced SDS-PAGE of various fractions obtainedfrom operation of the SP column of FIG. 4. The left and bottom sides ofFIG. 5B are marked as they are in FIG. 5A.

FIG. 6 is an image of reduced SDS-PAGE of various fractions obtained inpost column steps (see FIG. 7), namely fractions from the UF/DF step,the sterile filtration step, and from the ammonium sulfate precipitationstep. The left and bottom sides of FIG. 6 are marked as they are in FIG.5A.

FIG. 7 is a flow chart of a APF chromatographic botulinum toxinpurification process within the scope of the present invention.

FIG. 8 is a graph comparing the effect of a soy protein concentration ona botulinum toxin type A complex production in an APF fermentationprocess, where the fermentation medium contained 1 wt % glucose and 1 wt% of a yeast extract. In FIG. 8 the X axis represents the weight percentconcentration in the fermentation medium of a particular hydrolyzed soyprotein (HySoy), the left side Y axis represents potency of the finalpurified botulinum toxin complex and the right side Y axis representsthe percent of cell lysis completed, as determined by the equation:

${{Cell}\mspace{14mu} {Lysis}\; (\%)} = {\frac{{OD}_{600\max} - {OD}_{{endpo}\mspace{11mu} {int}}}{{OD}_{\max}} \times 100}$

where OD_(600 max) corresponds to the optical density measured at 600 nmat the time of maximum growth, and OD_(600 endpoint) is at the time offermentation harvest.

DESCRIPTION

The present invention is based upon the discovery that a Clostridialtoxin can be purified by use of an animal product fee (APF) system andprocess. The present invention encompasses a animal product free systemand process for purifying a Clostridium botulinum neurotoxin. TheClostridium botulinum neurotoxin can be a botulinum toxin type Acomplex, such as a 300 kD, 500 kD or 900 kD (approximate molecularweights) complex or mixtures thereof. The Clostridium botulinumneurotoxin can be any one of the serotypes A, B, C, D, E, F or G ormixtures thereof. Additionally, the system and process can be practicedin conjunction with a recombinant, hybrid, chimeric or modifiedbotulinum toxin (light chain, heavy chain, or both chains together).

Significantly, the system and process disclosed herein is scalable,meaning that it can be used to purify the quantities of botulinum toxinobtained from an industrial or commercial process, as use forpharmaceutical production. Further, the system and process is also CGMP(certified good manufacturing practices) compliant, as required by theU.S. CFR (United States code of federal regulations), meaning that itcan comply with regulatory requirements.

Through experimentation, there were developed APF systems and processesto purify a Clostridial toxin, such as a Clostridium botulinum type A(Hall strain) neurotoxin complex. The Clostridial toxin is purified fromthe fermentation medium resulting from either a Schantz (non-APF)fermentation process or from an APF fermentation process. Schantzprocesses use animal derived products. Significantly, while an APFfermentation process can reduce or eliminate animal derived products(such as casein and meat broth) as nutrients from the media used toculture and ferment Clostridial bacteria, APF fermentation processes aretypically followed by one or more purification steps which make use ofanimal derived products, such as the enzymes DNase and RNasePurification of the fermentation medium is required to obtain bulkClostridial toxin. Bulk Clostridial toxin (pure toxin or toxin complex)can be used for compounding a Clostridial toxin pharmaceuticalcomposition.

Preferably, the present invention is practiced in conjunction with anAPF fermentation process. Practicing the present invention inconjunction with an APF fermentation process provides a combined APFfermentation process and an APF purification process. Additionally,systems and method of the present invention are optimized for operationupon an APF fermentation medium, as opposed to a casein or other animalprotein based fermentation medium. Practicing the presently inventionupon a non-APF fermentation can result in a lower yield and/or a lowerpotency of the purified botulinum toxin obtained.

Thus, although both the Schantz and APF botulinum toxin purificationprocesses use animal derived products such as benzamidine to stabilizethe botulinum toxin and DNase and RNase to remove nucleic acids presentwith the botulinum toxin in the fermentation medium (see e.g. Examples 6and 7), our invention permits a botulinum toxin can be purified withoutusing such animal derived products.

The present invention encompasses systems and processes for purifying aClostridial toxin, such as a botulinum toxin complex. Typically aparticular system within the scope of the present invention is operatedin conjunction with a particular process within the scope of the presentinvention. A system within the scope of the present invention cancomprise a plurality (preferably as a consecutive series) ofchromatography steps. A process within the scope of the presentinvention can comprise passing a Clostridial toxin fermentation mediumthrough the plurality of chromatography columns to thereby obtain ahighly purified and highly potent Clostridial toxin. Such a purifiedClostridial toxin is suitable for compounding a Clostridial toxinpharmaceutical composition. Important parameters of systems andprocesses within the scope of the present invention include theparticular columns, buffers and operating (column running) conditionsused.

A first broad step of In a particular embodiment of the invention can beto load a fermentation medium clarified culture onto a hydrophobicinteraction column (such as a Butyl Sepharose Fast Flow [“FF”] column).This first column captures the Clostridial toxin (such as a botulinumtoxin complex) and allows impurities to flow through the column. It wasfound that a hydrophobic interaction column provided an efficientcapture of a botulinum toxin complex (a large protein with a particulartertiary and quaternary structure) from fermentation medium withretention of the biological activity of the botulinum toxin complex,while also separating (flow through) of many impurities present with thebotulinum toxin in the fermentation medium. A suitable buffer is used toelute the captured (bound) Clostridial toxin from the hydrophobicinteraction column.

In a second broad step in a particular embodiment of the presentinvention, the eluent from the first column is loaded onto a secondcolumn to further purify the Clostridial toxin. It was found thatpreferably, if second column [provides a different mechanism forseparation of Clostridial toxin from impurities, then a second columnchromatography step can provide a further efficient purification step.Thus, preferably, the second chromatography step entails use of adifferent column, such as a SP Sepharose high performance [“HP”] column.

In post chromatography (column) steps eluent from the second column canthen be further processed to obtain highly purified bulk botulinum toxincomplex. These additional processing steps can include buffer exchangeby ultrafiltration and diafiltration, sterile filtration and preparationof an ammonium sulphate suspension of the purified botulinum toxincomplex.

Our invention encompasses a scalable and cGMP compliant system andprocess for purifying a botulinum toxin, which can result in obtaining abulk botulinum toxin with the characteristics set forth in Table 1.

TABLE 1 Purified Botulinum Neurotoxin Characteristics Appearance Whiteto off-white suspension Concentration 2.0-3.6 mg/ml Nucleic Acids(A260/A278) Not more than 0.6 Specific Potency 2.4-5.9 × 10⁷ (MLD50unit/mg) Immunological Identity Pass SDS-PAGE Conformed to standardSEC-HPLC 900 kDa toxin complex >95% total peak

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® has the characteristics set forth inTable 1 above. BOTOX® consists of a purified botulinum toxin type Acomplex, human serum albumin, and sodium chloride packaged in sterile,vacuum-dried form. The botulinum toxin type A is made from a culture ofthe Hall strain of Clostridium botulinum grown in a medium containingN-Z amine casein and yeast extract (i.e. non-APF process). The botulinumtoxin type A complex is purified from the culture solution by a seriesof precipitation (including acid precipitation) steps to a crystallinecomplex consisting of the active high molecular weight toxin protein andan associated hemagglutinin protein. The crystalline complex isre-dissolved in a solution containing saline and albumin and sterilefiltered (0.2 microns) prior to vacuum-drying. BOTOX® can bereconstituted with sterile, non-preserved saline prior to intramuscularinjection. Each vial of BOTOX® contains about 100 units (U) ofClostridium botulinum toxin type A complex, 0.5 milligrams of humanserum albumin and 0.9 milligrams of sodium chloride in a sterile,vacuum-dried form without a preservative.

To reconstitute vacuum-dried BOTOX® sterile normal saline without apreservative (0.9% Sodium Chloride injection) is used by drawing up theproper amount of diluent in the appropriate size syringe. Since BOTOX®is denatured by bubbling or similar violent agitation, the diluent isgently injected into the vial. It has been reported that BOTOX® has beenadministered thirty or more days after reconstitution with little lossof potency. During this time period, reconstituted BOTOX® is stored in arefrigerator (2° to 8° C.). Reconstituted BOTOX® is clear, colorless andfree of particulate matter. The vacuum-dried product is stored in afreezer at or below −5° C.

The present invention is based upon the discovery of media and processeswhich are free or substantially free of an animal product or an animalbyproduct useful for culture and fermentation of an organism (such as aClostridium botulinum bacterium) capable of producing biologicallyactive botulinum toxin. The botulinum toxin obtained can be used formaking botulinum toxin active ingredient pharmaceutical compositions.Thus, growth media are disclosed herein which have significantly reducedlevels of meat or dairy by-products and preferred media embodiments aresubstantially free of such animal products.

The present invention encompasses the surprising finding thatanimal-based products are not required in media for growth ofClostridium botulinum, and particularly that vegetable-based productscan replace animal-based products typically employed in such media forthe growth of Clostridium botulinum.

Media that are in current use for growth and fermentation of bacteriausually comprise one or more animal derived ingredients, such as cokedmeat. In accordance with the present invention, preferred media forgrowth of Clostridium botulinum contain anima derived ingredients whichcomprise no more than about five to about ten percent of the totalweight of the media. More preferably, media within the scope of theinvention comprise no more than about one to less than about fivepercent of the total weight of the media of anima-derived products. Mostpreferably, all media and cultures used for the growth of Clostridiumbotulinum for the production of botulinum toxin are completely free ofanimal derived products. These media include but are not limited tomedia for small and large scale fermentation of Clostridium botulinum,media for growth of cultures of Clostridium botulinum used to inoculatethe seed (first) media and fermentation (second) media, as well as andmedia used for long-term storage of cultures of Clostridium botulinum(e.g. stock cultures).

In certain preferred embodiments of the invention, the media for thegrowth of Clostridium botulinum and production of botulinum toxin cancomprise soy based products to replace animal derived products.Alternately, instead of a soy based product there can be used debitteredseed of Lupinus campestris It is known the protein content of L.campestris seed is very similar to that of soybean. Preferably, thesemedia include soybean or of L. campestris derived products that arehydrolyzed and that are soluble in water. However, insoluble soy or ofL. campestris products can also be used in the present invention toreplace animal products. Common animal derived products which can besubstituted by soy or of L. campestris products include beef heartinfusion (BHI), animal derived peptone products, such as Bacto-peptone,hydrolyzed caseins, and dairy by-products such as animal milk.

Preferably media containing soy-based or of L. campestris based productsfor the growth of Clostridium botulinum are similar to commonly usedgrowth media containing animal derived products except thatsubstantially all animal-derived products are replaced withvegetable-derived products. For example, soy based fermentation mediacan comprise a soy based product, a source of carbon such as glucose,salts such as NaCl and KCl, phosphate-containing ingredients such asNa₂HPO₄ KH₂PO₄, divalent cations such as iron and magnesium, ironpowder, and amino acids such as L-cysteine and L-tyrosine. Media used togrow cultures of Clostridium botulinum for inoculation (i.e. the seed orfirst medium) of the fermentation (second) media preferably contain atleast a soy based product, a source of salt such as NaCl, and a carbonsource such as glucose.

The present invention provides a method for the growth of Clostridiumbotulinum that maximizes the production of a botulinum toxin using mediathat are substantially free of animal-derived products. Growth ofClostridium botulinum for production of botulinum toxin can take placeby fermentation in media containing soy by-products that replaceingredients derived from animal by-products. The inoculant for thefermentation medium can be derived from a smaller scaled growth medium(a seed medium). Depending on the size and volume of the fermentationstep, the number of successive growths in seed media to increase thebiomass of the culture can vary. To grow a suitable amount ofClostridium botulinum for inoculating the fermentation medium, one stepor multiple steps involving growth in a seed medium can be performed.For a method of growing Clostridium botulinum that is free of animalderived products, it is preferable that growth of Clostridium botulinumoriginates from a culture stored in non animal derived media. The storedculture, preferably lyophilized, is produced by growth in mediacontaining proteins derived from soy and lacking animal by-products.Growth of Clostridium botulinum in a fermentation medium can take placeby inoculation directly from a stored, lyophilized culture.

In a preferred embodiment of the present invention, growth ofClostridium botulinum proceeds in two phases-seed growth andfermentation. Both of these phases are carried out in anaerobicenvironments. The seed growth phase is generally used to “scale-up” thequantity of the microorganism from a stored culture. The purpose of theseed growth phase) is to increase the quantity of the microorganismavailable for fermentation. In addition, the seed growth phase allowsrelatively dormant microbes in stored cultures to rejuvenate and growinto actively growing cultures. Furthermore, the volume and quantity ofviable microorganisms used to inoculate the fermentation culture can becontrolled more accurately from an actively growing culture than from astored culture Thus, growth of a seed culture for inoculation of thefermentation medium is preferred. In addition, any number of consecutivesteps involving growth in seed media to scale-up the quantity ofClostridium botulinum for inoculation of the fermentation medium can beused. It is noted that growth of Clostridium botulinum in thefermentation phase can proceed directly from the stored culture bydirect inoculation.

In the fermentation phase, a portion of a seed medium or all of a seedmedium containing Clostridium botulinum from the seed growth is used toinoculate a fermentation medium. Preferably, approximately 2-4% of aseed medium having Clostridium botulinum from the seed growth phase isused to inoculate the fermentation medium. Fermentation is used toproduce the maximum amount of microbe in a large-scale anaerobicenvironment (Ljungdahl et al., Manual of industrial microbiology andbiotechnology (1986), edited by Demain et al, American Society forMicrobiology, Washington, D.C. page. 84).

A botulinum toxin can be isolated and purified using methods of proteinpurification well known to those of ordinary skill in the proteinpurification art. See e.g. Coligan et al. Current Protocols in ProteinScience, Wiley & Sons; Ozutsumi et al. Appl. Environ. Microbiol. 49;939-943:1985.

For production of botulinum toxin, cultures of Clostridium botulinum canbe grown in a seed medium for inoculation of the fermentation medium.The number of successive steps involving growth in a seed medium canvary depending on the scale of the production of botulinum toxin in thefermentation phase. However, as previously discussed, growth in thefermentation phase may proceed directly from inoculation from a storedculture. Animal-based seed media generally are comprised of BHI,bacto-peptone, NaCl, and glucose for growth of Clostridium botulinum. Aspreviously discussed, alternative seed media may be prepared inaccordance with the present invention in which animal-based componentsare substituted with non-animal-based components. For example butwithout limitation, soy-based products can substitute for BHI andbacto-peptone in the seed medium for growth of Clostridium botulinum andproduction of botulinum toxin. Preferably, the soy-based product issoluble in water and comprises hydrolyzed soy, although cultures ofClostridium botulinum can grow in media containing insoluble soy.However, levels of growth and subsequent toxin production are greater inmedia derived from soluble soy products.

Any source of soy-based products may be used in accordance with thepresent invention Preferably, the soy is hydrolyzed soy and thehydrolyzation has been carried out using non-animal enzymes. Sources ofhydrolyzed soy are available from a variety of commercial vendors. Theseinclude but are not limited to Hy-Soy (Quest International), Soy peptone(Gibco) Bac-soytone (Difco). AMISOY (Quest), NZ soy (Quest), NZ soy BL4,NZ soy BL7, SE50M (DMV International Nutritionals, Fraser, N.Y.), andSE50MK (DMV). Most preferably, the source of hydrolyzed soy is Hy-Soy orSE50MK. Other potential sources of hydrolyzed soy are known.

Concentrations of Hy-Soy in the seed medium in accordance with thepresent invention range between 25-200 g/L Preferably, the concentrationof Hy-Soy in the seed medium ranges between 50-150 g/L Most preferablythe concentration of Hy-Soy in the seed medium is approximately 100 g/L.In addition, the concentration of NaCl ranges between 0.1-2.0 g/L.Preferably the concentration of NaCl ranges between 0.2-1.0 g/L. Mostpreferably, the concentration of NaCl in the seed medium isapproximately 0.5 g/L. The concentration of glucose ranges between 0.1g/L and 5.0 g/L. Preferably, the concentration of glucose ranges between0.5-2.0 g/L. Most preferably, the concentration of glucose in the seedmedium is approximately 1.0 g/L. It is also preferred but not necessaryfor the present invention that the glucose is sterilized by autoclavingtogether with the other components of the seed medium. The pH level ofthe seed medium prior to growth can be 7.5-8.5. For example, the pH ofthe seed medium prior to growth of Clostridium botulinum can beapproximately 8.1.

Growth of Clostridium botulinum in the seed medium can proceed in one ormore stages. Preferably, growth in the seed medium proceeds in twostages. In stage one, a culture of Clostridium botulinum is suspended ina quantity of seed medium and incubated at 34±1° C. for 24-48 hours inan anaerobic environment. Preferably, growth in stage one proceeds forapproximately 48 hours. In stage two, a portion or all of the stage onemedium containing Clostridium botulinum is used to inoculate a stage twoseed medium for further growth. After inoculation, the stage two mediumis incubated at 34±1° C. for approximately 1-4 days also in an anaerobicenvironment. Preferably, growth in the stage two seed medium proceedsfor approximately 3 days. It is also preferable that growth in seedmedia in any stage does not result in cell lysis before inoculation offermentation media with the final growth in seed medium.

Standard fermentation media containing animal by-products for the growthof Clostridium botulinum can be based on a recipe of Mueller and Miller(MM; J. Bacteriol. 67.271, 1954). The ingredients in MM media containinganimal by-products include BHI and NZ-CaseTT. NZ-CaseTT is acommercially available source of peptides and amino acids which arederived from the enzymatic digestion of caseins, a group of proteinsfound in animal milk. The present invention demonstrates that non-animalbased products may be substituted for BHI and NZ-CaseTT in fermentationmedia. For example but without limitation, soy-based products canreplace the animal-based components of MM media used for fermentation ofClostridium botulinum. Preferably, the soy-based products arewater-soluble and derived from hydrolyzed soy, although as previouslydiscussed, insoluble soy products can also be used to practice thepresent invention.

Any source of soy-based products may be used in accordance with thepresent invention. Preferably, the hydrolyzed soy is obtained from QuestInternational (Sheffield) under the tradename, Hy-Soy or from DMVInternational Nutritionals (Fraser, N.Y.) under the tradename, SE50MKSoluble soy products can be also obtained from a variety of sourcesincluding but not limited to Soy peptone (Gibco) Bac-soytone (Difco),AMISOY (Quest), NZ soy (Quest), NZ soy BL4, NZ soy BL7, and SE50MK (DMVInternational Nutritionals, Fraser, N Y.).

In another preferred embodiment of the present invention, the mediumused for fermentation of Clostridium botulinum is free of animalby-products and comprises hydrolyzed soy, glucose. NaCl, Na₂HPO,MgSO₄7H₂. KH₂PO₄, L-cysteine, L-tyrosine, and powdered iron. Asdisclosed for the seed medium, hydrolyzed soy can replace animalby-products in fermentation medium. These animal by-products include BHIand NZ-Case TT (enzymatically digested casein).

The concentration of Hy-Soy in the fermentation medium for production ofbotulinum toxin preferably ranges between approximately 10-100 g/L.Preferably, the concentration of Hy-Soy ranges between approximately20-60 g/L. Most preferably, the concentration of Hy-Soy in thefermentation medium is approximately 35 g/L. For maximal production ofbotulinum toxin, particularly preferred concentrations of components inthe fermentation medium are approximately 7.5 g/L, glucose; 5.0 g/LNaCl; 0.5 g/L Na₂HPO₄; 175 mg/L KH₂PO₄; 50 mg/L MgSO₄7H₂O; 125 mg/LL-cysteine; and 125 mg/L L-tyrosine. The amount of powdered iron usedcan range from 50 mg/L to 2000 mg/L. Preferably, the amount of powderediron ranges between approximately 100 mg/L and 1000 mg/L. Mostpreferably, the amount of powdered iron used in fermentation mediaranges between approximately 200 mg/L and 600 mg/L.

For optimal levels of toxin production, the initial pH (beforeautoclaving) of the soy-based fermentation media ranges preferablybetween approximately 5.0 to 7.1. We found that pH control improvesbotulinum toxin recovery. Preferably the initial pH of the fermentationmedium is about pH 7. As explained in Example 7, we have found that ahigh yield of stable botulinum toxin can be obtained if the pH isthereafter reduced to and maintained between pH 5-5.5. As described forthe seed medium, the components of the fermentation medium, includingglucose and iron, are preferably autoclaved together for sterilization.

Preferably, a portion of the second stage seed medium used for growth ofClostridium botulinum is used to inoculate the fermentation medium.Fermentation occurs in an anaerobic chamber at approximately 34±1° C.for approximately 7 to 9 days. Bacterial growth can be monitored bymeasuring the optical density (O.D.) of the medium. Fermentationpreferably is stopped after cell lysis has proceeded for at least 48hours as determined by growth measurement (optical density). As cellslyse, the O.D. of the medium decreases.

In a preferred embodiment of the present invention, cultures ofClostridium botulinum used for long-term storage of Clostridiumbotulinum and inoculation of the seed medium are grown and lyophilizedin soy-milk prior to storage at 4° C. Cultures of Clostridium botulinumin animal milk lyophilized for storage can also be used for theproduction of botulinum Toxin. However, to maintain media that aresubstantially free of animal by-products throughout the production ofbotulinum toxin, it is preferred that the initial culture of Clostridiumbotulinum be preserved in soy milk and not animal milk.

EXAMPLES

The following examples set forth specific methods encompassed by thepresent invention and are not intended to limit the scope of theinvention. Unless explained otherwise in these Examples “toxin” or“botulinum toxin” means a botulinum toxin type A complex with amolecular weight of about 900 kDa. Our invention is not limited tosystems and method for purifying a botulinum toxin type A complex with amolecular weight of about 900 kDa, having ready applicability to thepurification of 150 kDa, 300 kDa, 500 kDa and well as other molecularweight toxins, complexes and botulinum toxin serotypes.

Example 1 Preparation of an Animal Product Free Seed Medium forClostridium Botulinum

A control seed medium can be prepared using the following ingredientsfor each one 1 liter of medium. NaCl (5 g), Bacto-peptone (10 g),glucose (10 g), BHI (to 1 liter), pH 8.1 (adjusted with 5 N NaOH).

A test (animal product free) seed medium can be prepared using thefollowing ingredients for each one 1 liter of medium: NaCl (5 g),Soy-peptone (10 g), glucose (10 g), Hy-Soy (35 g/liter, to make up 1liter of media fluid), pH 8.1 (adjusted with 5 N NaOH).

Example 2 Culturing Clostridium Botulinum in an Animal Product Free SeedMedium

A lyophilized culture of the Clostridium botulinum can be suspended in 1ml of each of the control and test seed medium of Example 1, divided(each seed media) into two tubes of which each can contain 10 ml of therespective seed media, and then incubated at 34° C. for about 24-48hours. One ml of culture can be then used to inoculate a 125 ml DeLongBellco Culture Flask containing 40 ml of (the respective) seed media.The inoculated culture can be incubated at 33° C. 1° C. for 24 hours ina Coy Anaerobic Chamber (Coy Laboratory Products Inc., Grass Lake,Mich.).

Example 3 Preparation of an Animal Product Free Fermentation Media forClostridium Botulinum

A basal fermentation medium can be prepared using the followingingredients for each two liters of medium: glucose (15 g), NaCl (10 g),NaH₂PO₄ (1 g), KH₂PO₄ (0.350 g), MgSO₄7H₂O (0.1 g), cysteine-HC (0.250g), tyrosine-HCl (0.250 g), powdered iron (1 g), ZnCl₂ (0.250 g), andMnCl₂ (0.4 g).

A control fermentation medium can be prepared using the followingingredients for each two liters of medium prepared: BHI (500 ml: thiscorresponds to about 45.5 grams of dry weight beef heart infusion),NZ-CaseTT (30 g), and basal medium (to 2 liters), pH 6.8.

The basal fermentation medium can be prepared first and adjusted to pH6.8. The beef heart infusion (BHI) BHI can then be prepared and its pHadjusted to 0.8 with 5 N NaOH. The BHI can then be added to the basalmedium. Next the NZ-CaseTT can be prepared. The NZ-Case TT is then addedto the to basal medium to which the beef heart infusion has already beenadded, and dissolved by addition of HCl. The pH can then be adjusted to6.8 with 5 N NaOH. This medium can then be separated into 8 ml portionsinto each of sixteen 100 mm test tubes, following by autoclaving for 25minutes at 120° C.

A test fermentation medium (animal product free) can be prepared bysubstituting a test nitrogen source for the BHI present in the controlfermentation medium. Suitable test fermentation medium nitrogen sourcesinclude: Hy-Soy (Quest), AMI-Soy (Quest), NZ-Soy (Quest), NZ-Soy BL4(Quest), NZ-Soy BL7 (Quest). Sheftone D (Sheffield), SE50M (DMV), SE50(DMV), SE %)MK (DMV), Soy Peptone (Gibco), Bacto-Soyton (Difco),Nutrisoy 2207 (ADM), Bakes Nutrisoy (ADM) Nutrisoy flour, Soybean meal,Bacto-Yeast Extract (Difco) Yeast Extract (Gibco), Hy-Yest 412 (Quest),Hy-Yest 441 (Quest), Hy-Yest 444 (Quest), Hy-Yest (455 (Quest)Bacto-Malt Extract (Difco), Corn Steep, and Proflo (Traders).

The test fermentation medium can be prepared as set forth above for acontrol fermentation medium except that BHI is excluded and the relevantnitrogen source can be first adjusted to pH 6.8 with 3 N HCl or with 5 NNaOH. The media can be allocated to in 8 ml portions to sixteen 100 mmtest tubes, followed by autoclaving for 20-30 minutes at 120° C.

Example 4 Growth of Clostridium Botulinum in an Animal Product FreeFermentation Medium

A 40 μl portion of the test seed medium culture (animal product free)can be used to inoculate each 8 ml control or test fermentation mediumaliquot in an 8 ml 16×100 mm test tube. The cultures can then beincubated at 33±1° C. for 24 hours. Tubes can then be incubated in ananaerobic chamber to allow for growth of the bacterium. Each mediumassay can be performed in triplicate (i e can involve three independentinoculations of the same medium), and can also include a non-inoculatedcontrol, which can be used as the blank for the spectrophotometer).Growth (as determined by optical density, OD) can be measured every 24hours with a Turner Spectrophotometer (Model 330) at 660 nm. Cultivationshould be stopped after cell lysis has lasted for about 48 hours andbotulinum toxin production can then be measured.

Additional experiments can be carried out with a Hy-Soy fermentationmedium containing the following ingredients for each 500 ml of themedium: Hy-Soy (17.5 g), glucose (3.75 g); NaCl (2.5 g); Na₂HPO₄ (0.25g), MgSO₄7H₂O (0.025 g), KH₂PO₄ (0.0875 g), L-cysteine (0.0625 g),L-tyrosine (0.0625 g), powdered iron (0.25 g), pH 6.8

Example 5 Determination of Botulinum Toxin Production by ClostridiumBotulinum Grown in an Animal Product Free Fermentation Medium

The cultured cells of Example 4 can be centrifuged, and the pH of thesupernatant then determined. The levels of botulinum toxin in a givensample can be measured by adding a standard antitoxin and measuring theelapsed time before flocculation. Both Kf (the time required forflocculation to occur, in minutes) and Lf (the limit of flocculation;equivalent to 1 international unit of standard antitoxin, as establishedby flocculation) can be determined. 4 ml of fermentation broth can betaken from each fermentation tube for a given culture, and can becombined together so that 12 ml total can be mixed in a 15 ml centrifugetube. The tubes can be centrifuged at 5000 rpm (3400 g) for 30 min at 4°C. 1 ml aliquots of supernatant can be added to tubes containing 0.1-0.6ml of standard botulinum toxin antiserum, and the tubes can be carefullyshaken to mix their contents. The tubes can then be placed in a waterbath at 45±1° C. and the initial time can be recorded. The tubes can bechecked frequently, and the time at which flocculation began can berecorded as Kf. The concentration of toxin in the tube in whichflocculation can be first initiated can be designated LfFF. Theconcentration of toxin the tube in which flocculation can be initiatedsecond can be designated LfF.

Parallel fermentation, growth and toxin production assays can be carriedout for both of (a) the control seed medium (used to inoculate thecontrol fermentation medium) and the control fermentation medium, and;(2) the (animal product free) test seed medium (used to inoculate thetest fermentation medium) and the (animal product free) testfermentation medium. Significantly, it can be determined that thefermentation of Clostridium botulinum in media free of animal productsand inoculated from cultures also free of animal products (with soy-baseproducts replacing the animal products) can result in an Lf_(toxin) ofapproximately 50 or more. Minimally, Lf_(toxin) equals approximately 10.Preferably the Lf_(toxin) is at least 20. Most preferably the Lf_(toxin)is greater than 50.

Additionally, it can be determined that various soy products supportClostridium botulinum growth in fermentation media lacking BHI. Thussoluble soy preparations can replace BHI for growth of Clostridiumbotulinum. The best concentration can be 125 or 25 g/L. Hy-Soy(Sheffield) can give the highest growth. Insoluble soy preparations canbe less effective.

Furthermore, results can be obtained to show that Quest Hy-Soy, DMVSE50MK, and Quest NZ-Soy can be effective soy products in terms of theirability to replace BHI for Clostridium botulinum growth. The results canreveal that the soy products (such as Quest Hy-Soy. DMV SE50MK, andQuest NZ-Soy) that may be optimal for growth can also be effective atreplacing BHI for toxin production. The best soy product for toxinproduction can be Quest Hy-Soy at 22.75 g/l. Higher concentrations ofthis product may produce better growth but not improve toxin production.Similar results can, it is proposed, be obtained with SE50MK, for whicha higher concentration may generate increased growth, but not increasetoxin production. NZ-Soy, on the other hand, may give higher growth andhigher toxin production at its higher concentration.

Finally, it can be determined that soy products can effectively replaceBHI as well as the NZ-CaseTT. Removal of NZ-CaseTT from soy-based mediacan reduce growth of about 2-4 fold. The best soy product for growthboth in the presence and the absence of NZ-CaseTT can be SE50MK. HY-Soycan replace both BHI and NZ-CaseTT for toxin production. However, alonger fermentation cycle of 1 or 2 days may be necessary. HY-Soy couldreplace both BHI and NZ-CaseTT in media for toxin production. However,it can be determined that yeast extracts can be inhibitory to toxinproduction.

It can be determined that HY-Soy at 22.75 g/l may completely replaceboth BHI and HY-CaseTT for toxin production. Unlike the effect on growthwhere 56.88 g/l HY-Soy can be best, 34.13 g/HY-Soy can be best for thetoxin production phase.

Thus, it has surprisingly been determined if Hy-Soy or [Hy-Soy+Hy-Yest]can replace BHI and Bacto-peptone in media for seed growth ofClostridium botulinum. In addition, experiments can be designed todetermine the optimum concentrations of components in seed media toproduce the maximum levels of botulinum toxin production by theClostridium botulinum. Toxin production by Clostridium botulinum grownin seed medium and fermentation medium that is free of BHI and NZ-CaseTTcan reach or exceed levels attained in media containing BHI andNZ-CaseTT.

It can be determined that the optimum concentrations of Hy-Soy or[Hy-Soy+Hy-Yest] for growth in the seed medium. Experiments can confirmthat Hy-Soy can replace BHI and Bacto-peptone as the nitrogen source inseed medium for growth of Clostridium botulinum and for production ofbotulinum toxin in the subsequent fermentation phase. Also, Hy-Soy asnitrogen source in the seed medium, as compared to Hy-Soy plus Hy-Yest,can produce higher levels of botulinum toxin in the subsequentfermentation step. The concentrations of Hy-Soy in seed medium thatproduce the best levels of toxin range from approximately 62.5 g/L to100 g/L.

Additional experiments can be designed to determine the optimumconcentrations of Hy-Soy in the seed medium for the maximum productionof botulinum toxin by Clostridium botulinum by fermentation. Thus, 30 g,50 g, 75 g and 100 g of Hy-Soy in the seed medium can all resulted inproduction of botulinum toxin by fermentation of Clostridium botulinumand this is comparable or exceeds levels of botulinum toxin made in seedmedium containing BHI and Bacto-peptone as a nitrogen source.

It can be found that a concentration of 100 g/L Hy-Soy in the seedmedium resulted in the highest levels of toxin production in thesubsequent fermentation step. In addition, the data indicate that seedstep-1 of Hy-Soy seed medium produced greater growth after 48 hours thanafter 24 hours.

Example 6 Non-APF Process for Obtaining a Botulinum Toxin

A Clostridial toxin was obtained by fermentation of a Clostridiumbotulinum bacterium. Thus, a modified Schantz (non-APF) process wascarried out to obtain highly potent and highly purified Clostridiumbotulinum toxin (i.e. bulk toxin) as follows. A modified Schantz(non-APF) process can provide a high yield of botulinum toxin. BothSchantz and modified Schantz processes use casein in all thefermentation media.

Stock Culture Preparation

Various Clostridial bacteria are available from the American TypeCulture Collection (ATCC), Manassas, Va. Alternately, a Clostridiumbotulinum cell bank vial can be prepared by isolating Clostridiumbotulinum from various sources, including soil or by deep sampling (atanaerobic or at quasi-anaerobic locations) of putrefying animalcarcasses. Commonly, Clostridium botulinum can be obtained from a sampleof a physiological fluid (i.e. a wound swap from a patient with woundbotulism) of a patient diagnosed with botulism. The top half of FIG. 1summarizes the non-APF process used for preparation of a cell bank vial,and for the culture and fermentation of a botulinum toxin.

The Clostridium botulinum obtained from a natural or patient source iscultured on blood agar plates, followed by inoculation of high growthcolonies into a cell bank vial medium. The cell bank vial medium usedfor Clostridium botulinum was a cooked meat medium which containschopped fresh beef. Actively growing cultures were mixed with glycerolto prepare a cell bank vial (i.e. a stock culture) of the Clostridiumbotulinum bacterium which was frozen for later use.

Seed Cultivations

A Clostridium botulinum cell bank vial was thawed at room temperature,followed by four cultivation steps. (1) To select colonies with asuitable morphology, aliquots from the thawed cell bank vial werecultivated by streaking the bacterium on pre-reduced Columbia blood agarplates and anaerobically incubating for 30-48 hours at 34° C.±1°. (2)Selected colonies were then inoculated into test tubes containing acasein growth medium for 6-12 hours at 34° C. The contents of the tubewith the most rapid growth and highest density (growth selection step)were then further cultivated through two step-up anaerobic incubations:(3) a first 12-30 hour incubation at 34° C. in a one liter seedcultivation bottle, followed by (4) a second cultivation in a 25 literseed fermenter containing a casein growth medium for 6-16 hours at 35′C. These two step-up cultivations were carried out in a nutritive mediacontaining 2% casein hydrolysate (a casein [milk protein] digest), 1%yeast extract and 1% glucose (dextrose) in water at pH 7.3.

Fermentation

The step-up cultivations were followed by a further incubation for 60-96hours at 35° C. in a commercial scale (i.e. 115 liter) fermenter in acasein containing medium under a controlled anaerobic atmosphere. Growthof the bacterium is usually complete after 24 to 36 hours, and duringthe 60-96 hour fermentation most of the cells undergo lysis and releasebotulinum toxin. Control of the fermentation medium pH is not requiredin a Schantz or modified Schantz process. It is believed that toxin isliberated by cell lysis and activated by proteases present in theculture broth. Optionally, a filtration of this culture medium using asingle layer depth filter to remove gross impurities (i.e. whole andruptured cells) can be prepared to obtain a clear solution referred to aclarified culture,

Harvest

Harvest of toxin can be accomplished by lowering the pH to 3.5 withsulfuric acid to precipitate the raw toxin at 20° C. The raw toxin wasthen concentrated by ultramicrofiltration followed by diafiltration.

Purification

The harvested crude toxin was then transferred to a digestion vessel andstabilized by addition of the protease inhibitor benzamidinehydrochloride, DNase and RNase were added to digest nucleic acids. Thetoxin containing material was subjected to UF/DF and three precipitationsteps (cold ethanol, hydrochloric acid and ammonia sulfateprecipitations). The purified botulinum neurotoxin complex (bulk toxin)was stored as a suspension in a sodium phosphate/ammonium sulphatebuffer at 2-8 degrees C.

The resulting bulk toxin was a high quality crystalline 900 kD botulinumtoxin type A complex made from the Hall A strain of Clostridiumbotulinum with a specific potency of ≥3×10⁷ U/mg, an A₂₆₀/A₂₇₈ of lessthan 0.60 and a distinct pattern of banding on gel electrophoresis, andsuitable for use for the compounding of a botulinum toxin pharmaceuticalcomposition.

Compounding can encompass a many fold dilution of the bulk toxin, mixingwith one or more excipients (such as albumin and sodium chloride) tothereby form a toxin composition, and preparation of a storage andshipment stable form of the toxin composition, as by lyophilizing,freeze drying or vacuum drying the composition.

The purified botulinum toxin complex obtained from a Schantz or modifiedSchantz process can be eluted from an ion exchange column in a pH 7-8buffer to disassociate the non toxin complex proteins from the botulinumtoxin molecule, thereby providing (depending upon the type ofClostridium botulinum bacterium fermented) pure botulinum toxin type Awith an approximately 150 kD molecular weight, and a specific potency of1-2×10⁸ LD₅₀ U/mg or greater, or purified botulinum toxin type B with anapproximately 156 kD molecular weight and a specific potency of 1-2×10⁶LD₅₀ U/mg or greater, or purified botulinum toxin type F with anapproximately 155 kD molecular weight and a specific potency of 1-2×10⁷LD₅₀ U/mg or greater.

As set forth supra, in one aspect our invention eliminates the harvestpurification steps set forth in this Example 6 carried out uponclarified culture, including elimination of use of the animal derivedproducts, such as RNase and DNase.

Example 7 APF Media and Process for Obtaining a Botulinum Toxin

This Example 7 sets forth an APF process carried out to obtain highlypotent and highly purified Clostridium botulinum toxin type A (i.e. bulktoxin). The process can be used with other botulinum toxin serotypes.

Stock Culture Preparation

As set forth in Example 6. Clostridial botulinum can be obtained fromthe ATCC, from various sources in nature or from a botulism patient. Thebottom half of FIG. 1 summarizes the APF process used for preparation ofa cell bank vial, and for the culture and fermentation of a botulinumtoxin. APF cell bank vials were prepared by culturing Clostridiumbotulinum on plant agar plates. The plant agar plates were made bymixing the soy derivative HySoy (Quest) with a yeast extract and glucosein a 3:1:1 (weight percent) ratio with agar and allowing setting. Othercommercially available APF agar plates or dehydrated powder for makingthe plates were also found to be suitable. Selected high growth colonieswere then inoculated into an APF cell bank vial medium. The APF cellbank vial medium used comprised hydrolyzed soy protein, yeast extract(no animal product was used in either the cultivation of the yeast or inthe process for preparation of the yeast extract made therefrom) andglucose in the same 3:1:1 ratio. Other nutrient ratios (i.e. 6:1:1,6:0:1 and 6:3:1 were also found to be suitable). The hydrolyzed soy(HySoy) and yeast extract (HyYest) concentrates used were obtained fromQuest International. The Clostridium botulinum culture in the APF mediumwas combined with glycerol, aliquoted to cryovials and frozen for lateruse. The APF media developed can be used to store the Clostridialbotulinum bacteria for a period of one year or longer without loss ofviability. These frozen culture and glycerol mixtures in cryovials arethe APF cell bank vials.

Seed Cultivations

An APF cell bank vial was thawed at room temperature, followed by asingle cultivation step: a one liter seed culture bottle was theninoculated directly (i.e. without an intervening blood agar culture ortube growth steps) with the APF cell bank vial contents using the sameAPF medium (the APF cell bank vial [storage] medium can be differentfrom the APF fermentation [growth] medium) and maintained at 35° C. for15 to 24 hours, with an initial medium pH of 7.0 in an anaerobic(nitrogen) atmosphere.

Fermentation

Next the seed bottle culture was transferred to a commercial scale 10liter production fermenter containing the APF medium (hydrolyzed soyprotein, yeast extract and 1% glucose) maintained at 35° C. for 52-72hours, with an initial medium pH of 7.0, in an anaerobic (nitrogen)atmosphere. Approximately 15 hours after commencement of thefermentation (the culture pH has naturally decreased to below 6.0), a pHcontrol program at range of pH 5.0-5.5 was initiated by adding HCl tothe culture. It was found that it was necessary to control the pH of theAPF fermentation medium within the narrow range in order to obtain anacceptable yield of active botulinum toxin. Thus, it was found that thispH control to between pH 5.0-5.5 substantially prevented degradation andloss of potency of the botulinum toxin. It is believed that during thefermentation most of the cells undergo lysis and release botulinum toxinand that toxin liberated by cell lysis is activated by proteases presentin the culture broth Filtration of this culture medium using a singlelayer depth filter removes gross impurities (i.e. whole and rupturedcells) and results in a clear solution referred to a clarified culture.

Harvest

Harvest of botulinum toxin can then proceed as in Example 6 (i.e.sulfuric acid precipitation, followed by concentrated by microfiltrationfollowed by diafiltration).

Purification

Purification of the toxin can then proceed as set forth in Example 6:i.e. addition of benzamidine hydrochloride, and DNase and RNase,sulfuric acid precipitation, cold ethanol precipitation, phosphatebuffer extraction, hydrochloric acid precipitation, phosphate bufferextraction and bulk toxin storage.

As an alternative to the Example 6 harvest and purification process, acolumn chromatography process of the present invention can be carriedout.

The resulting bulk toxin is a high quality crystalline 900 kD botulinumtoxin type A complex made from the Hall A strain of Clostridiumbotulinum with a specific potency of ≥3×10⁷ U/mg, an A₂₆₀/A₂₇₈ of lessthan 0.60 and a distinct pattern of banding on gel electrophoresis, andsuitable for use for the compounding of a botulinum toxin pharmaceuticalcomposition. Thus, this APF process for a botulinum toxin can generatehigh quality toxin.

The purified botulinum toxin complex obtained from an APF process can bepassed through and eluted from an ion exchange column in a pH 7-8 bufferto disassociate the non toxin complex proteins from the botulinum toxinmolecule, thereby providing (depending upon the serotype of Clostridiumbotulinum bacterium fermented) botulinum toxin with an approximately 150kD molecular weight, and a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; or purified botulinum toxin type 8 with an approximately 156 kDmolecular weight and a specific potency of 1-2×10⁸ LD₅₀ U/mg or greater,or purified botulinum toxin type F with an approximately 155 kDmolecular weight and a specific potency of 1-2×10⁷ LD₅₀ U/mg or greater.For example, by use of our APF medium we were able to obtain a botulinumtoxin type A complex with a specific potency of 1.02×10⁸ LD₅₀ U/mg ofthe botulinum toxin.

In this Example 7 APF media with either 1% by wt or 2% by wt glucosewere used (note that 1% glucose means 1 g of glucose per 100 ml of theculture medium and 2% glucose means 2 g of glucose were present for each100 ml of the culture medium) and it was determined that maximalbacterium growth (as determined by peak optical density [optical densitywas measured at 600 nm] of the culture) occurred after about 20 hours offermentation in the 1% glucose APF medium vs after about 40 hours offermentation in the 2% glucose APF medium, but that the peak opticaldensities did not differ significantly as the glucose content of themedia was so varied. It was believed that cell autolysis and toxinrelease resulted in a maximal amount of active botulinum toxin in the 1%glucose APF media (as determined by a SNAP-25 assay for active toxin)after about 55 hours of fermentation, but that with the 2% glucose APFmedia the amount of active botulinum toxin present in the medium at alater time (as determined by a SNAP-25 assay for active toxin) and wasstill increasing after 65 hours of fermentation. Thus, a more rapidrelease of botulinum toxin occurred with use of the lower (1%) glucoseAPF medium amount present, indicating that a more efficient toxinproduction process (i.e. more amount of toxin obtained per unit of time)can be carried out with use of the lower (1%) glucose APF medium.

As shown by FIG. 1, it was also determined that optimal parameters forproduction of botulinum toxin in an APF medium were the combination ofthe following parameters: (1) about 6% by weight of a hydrolyzed soyconcentration (“HySoy Conc.” in FIG. 1) in the APF fermentation medium.6% soy means 6 g of the soy protein per 100 ml of the culture medium;(2) 0% to 3% yeast extract concentrate (“YE Conc.” In FIG. 1) in the APFfermentation medium; (3) 50-72 hours of fermentation at a temperature of33-35° C. under anaerobic (nitrogen atmosphere) conditions; (4) pH ofthe fermentation medium maintained between about pH 5.0 to 5.5throughout the fermentation period after the initial cell growth, and(5) 1 wt % glucose in the APF fermentation medium.

Thus, as shown by FIG. 1 as more protein is present in the APF medium(as the total amount of HySoy and YE) the pH of the medium tends toincrease with resulting lower toxin stability and that when the pH waslowered with the same total protein nutrient content in the medium,toxin production yield increased dramatically. In the non-APF processthe total protein content is lower so that pH does not tend to rise andtherefore there is no elevated pH to have a deleterious effect on toxinproduction. FIG. 1 shows that there was consistently more activity (asdetermined by the MLD50 and SNAP-25 assays) when the pH of the mediumwas controlled to within a narrow range of about 5.3 to 5.5. FIG. 1 alsoshows that the highest toxin yield (as determined by the SNAP 25 assay)was obtained with a medium which comprised 6% hydrolyzed soy and 1%yeast extract. FIG. 8 shows that when the yeast and glucose nutrientswere both at 1%, that cell lysis between 68-100% and potency as high asabout 38×105 units/mL of toxin was obtained, as soy protein was variedfrom 1 to 6% weight.

The SNAP-25 assay used was an ELISA based method to measure SNAP-25proteolytic activity of the botulinum toxin. SNAP-25 is an abbreviationfor synaptosome associated protein of 25 kDa molecular weight. SNAP-25is a 206 amino acid plasma membrane protein involved in neuronalexocytosis. The assay is based on the method disclosed in Ekong T., etal., Recombinant SNAP-25 is an effective substrate for Clostridiumbotulinum type A toxin endopeptidase activity in vitro. Microbiology(1997), vol 143, pages 3337-3347 The assay uses a truncated SNAP-25protein (the 206 amino acid residue peptide) bound to polystyrene 96well microtiter plates and a monoclonal antibody that recognizes thecleaved product (a 197 amino acid residue peptide) which is made byenzymatic hydrolysis between amino acids 197 and 198 of the SNAP-25 byreduced botulinum toxin type A. The monoclonal antibody bound to thecleaved product is then detected with a secondary antibody (goatanti-mouse IgG conjugated to horseradish peroxidase [HRP)], whichproduces a color change in the presence of a chromogenic substrate(TMB).

The MLD50 (mouse 50% lethal dose) assay is a method for measuring thepotency of a botulinum toxin by intraperitoneal injection of thebotulinum toxin into female mice (about four weeks old) weighing 17-22grams each at the start of the assay. Each mouse is held in a supineposition with its head tilted down and is injected intraperitoneallyinto the lower right abdomen at an angle of about 30 degrees using a 25to 27 gauge ⅜″ to ⅝″ needle with one of several serial dilutions of thebotulinum toxin in saline. The death rates over the ensuing 72 hours foreach dilution are recorded. The dilutions are prepared so that the mostconcentrated dilution produces a death rate of at least 80% of the miceinjected, and the least concentration dilution produces a death rate nogreater than 20% of the mice injected. There must be a minimum of fourdilutions that fall within the monotone decreasing range of the deathrates. The monotone decreasing range commences with a death rate of noless than 80%. Within the four or more monotone decreasing rates, thetwo largest and the two smallest rates must be decreasing (i.e. notequivalent) The dilution at which 50% of the mice die within the threeday post injection observation period is defined as a dilution whichcomprises one unit (1 U) of the botulinum toxin.

Significantly, the APF process of this Example 7 differs from theExample 6 non-APF process, by at least: (1) replacing the cell bank vialcooked meat medium with an APF medium: (2) eliminating the blood agarcolony selection step; (3) eliminating the subsequent casein mediumbased tube growth step, and; (4) replacing the non-APF fermentationmedia with APF media throughout.

FIG. 2 presents a summary of the differences between an industrial scale(non-APF) Schantz process (Example 6 and the industrial scale APFprocess of Example 7, through the cell bank creation, culture andfermentation steps. FIG. 2 omits the harvest and purification steps.

The APF media can be used to select for Clostridium botulinum bacteria.Thus, concurrent practice of the Examples 6 and 7 initial culture stepspermits isolation and growth of a Clostridium botulinum culture withcharacteristics conducive to growth and production of botulinum toxinsin or on an APF medium. The transfer of Clostridium botulinum bacteriafrom a non-APF medium to an APF medium enriches for and selects forbacteria that can either adapt to the new environment or throughselective die off of bacteria that cannot grow and produce in the newenvironment.

Example 8 Chromatographic Systems and Methods for Purifying a BotulinumToxin

The chemicals used in the experiments set forth in Examples 8 andfollowing included:

10N NaOH (Mallinckrodt, VWR Cat # MKH38505) Acetic Acid, USP/FCC Grade,99.5-100.5% (J. T. Baker, Cat # JT9522-2) Ammonium Sulfate, Ultrapure,99% (ICN, Cat # IC808211) Citric Acid, USP/FCC Grade, 99.5-100.5% (J. T.Baker, Cat # JT0119-1)

Ethanol, anhydrous, denatured (JT Baker, Cat #9299-1)Hydrochloric acid. NF/FCC Grade, 36.5-38%-Mallinckrodt-MK2612-14Phosphoric acid, NF/FCC, 85%-88% (Mallinckrodt, Cat # MK278814)Sodium acetate trihydrate, 99%-101%, USP/FCC (Mallinckrodt, Cat#MK735602)Sodium chloride, USP/FCC Grade, 99.0-101.0 (Mallinckrodt, Cat #MK753204)Sodium citrate, USP/FCC Grade, 99.0-100.5% (J. T. Baker, Cat #JT3650-1)Sodium hydroxide, NF/FCC Grade, 95.0-100.5%-Mallinckrodt-MK768004Sodium phosphate, dibasic Heptahydrate, USP (Mallinckrodt, Cat#MK789604)Sodium phosphate, monobasic monohydrate. USP/FCC (Mallinckrodt, Cat #MK786812)

The chromatography resins use in the experiments below included:

Bakerbond ABx Prepscale (JT Baker. Cat #7269-02)

Butyl Sepharose FF (Amersham Biosciences, Cat #17-0980-02)

Ceramic Hydroxyapatite, Type I (Bio-Rad, Cat #158-4000)

Ceramic Hydroxyapatite, Typer II (Bio-Rad, Cat #157-4200)

HiTrap HIC Selection Kit (Amersham Biosciences, Cat #17-1349-01)

HiTrap IEX Selection Kit (Amersham Biosciences. Cat #17-6002-33)

MEP Hypercel (Ciphergen, sample)

SP Sepharose HP (Amersham Biosciences, Cat #17-1087-03)

The equipment and accessories used is the experiments below included:

AKTA Purifier and AKTA FPLC Chromatography System (Amersham Biosciences)

Bottle-top 0.22 μm vacuum sterile filter (Nalgene)Labscale TFF system and Pellicon XL50 with Biomax 100 membrane(Millipore) (this is the ultrafiltration equipment).Masterflex US pump Model #77201-62 (Cole-Parmer)

Pellicon 2 Mini Holder (Millipore)

XK and HR columns (Amersham Biosciences)

The buffers used in our experiments are listed in Table 2.

TABLE 2 Buffers used in the APF purification process Purification StepsBuffers used Butyl Sepharose FF  1. 50 mM NaPi, 4M NaCl is pH6.0Chromatography  2. 50 mM NaPi, 2M NaCl is pH6.0  3. 50 mM NaPi, 1M NaClis pH6.0  4. 50 mM NaPi, pH 6.0 SP Sepharose HP  5. 20 mM Na citrate, pH4.0 Chromatography  6. 20 mM Na citrate, 300 mM NaCl, pH4.0  7. 20 mM Nacitrate: 400 mM NaCl, pH4.0  8. 20 mM Na citrate: 1M NaCl, pH4.0 PostPurification Steps Solutions used Post-column processes  9. 50 mM NaAc,pH 4.0 10. 25M ammonium sulfate Miscellaneous 11. 0.1N NaOH 12. 1N NaOH

In Table 2: buffers 1 and 2 were used to wash impurities off the column;buffers 3 and 4 was used to elute bound toxin from the column: buffer 5was used to dilute the eluent from the Butyl column buffer 6 was used towash impurities off the column; buffers 7 and 8 were used to elute boundtoxin from the column: buffer 8 was the UF/DF dialysis buffer; solution9 was used to precipitate toxin, and solutions 10 and 11 were used toinactivate (clean) any toxin remaining in the columns after use.

Example 9 Selection of Preferred Chromatography Columns for Use in anAPF Column Chromatographic Botulinum Toxin Purification (Capture Step)Process

This experiment established preferred chromatography columns andtechniques for initial purification of a botulinum toxin type A complexfrom attendant impurities in a fermentation medium.

Feed Materials

Both a filtered cell culture (clarified culture) obtained from an APFprocess fermentation and an extract thereof prepared by hydrochloricacid precipitation were assessed as chromatography column feedmaterials. It was found that direct loading of the clarified cultureonto a column prevented toxin precipitation and that a clarified culturefeed material was much easier to handle and validate. On the other hand,use as the feed material of a clarified culture extract prepared by acidprecipitation removed additional impurities and provided virusinactivation. With regard to the characteristics of process robustness,a clarified culture was determined to be the preferred feed material, asopposed to use of a hydrochloric acid precipitation preparation as thebulk botulinum toxin complex chromatography resin feed material. Hence,clarified culture was the preferred feed material.

Our studies showed that as the pH was lowered proteins (i.e. thebotulinum toxin complex) started to precipitate at about pH 5, thatsmall amounts of toxin was extracted (as most had precipitated out) atabout pH 4.0, and that essentially all of the toxin had precipitationout of the solution at between pH 3.5 to 3.8. On the other hand, wefound (based for example on SDS-PAGE and Western blotting) that mostimpurities were co-extracted with the botulinum toxin at a pH of 6.8.Hence, a preferred feed liquid pH for carrying out our purificationprocess invention was between about pH 5-6.8, with a more preferred pHbeing about pH 5.5 for extraction, that is separation of the botulinumtoxin from attendant impurities.

Capture Step

For the capture step botulinum toxin type A (Hall strain) cell culturefiltrates were incubated with a number of chromatography resins (seebelow) under the manufacturer specified conditions for use of eachparticular column.

After washing the columns, the column bound proteins were eluted withthe specified elution buffers. All eluted fractions were collected andanalyzed by SDS-PAGE. The results obtained (Table 3) were confirmed bychromatography using 1 ml HiTrap or HR5/5 columns.

TABLE 3 Summary of Capture Step Results Toxin Separation Toxin in inSeparation Technique Resin Flowthru Eluate Observed Hydrophobic PhenylFF − + + Interacton (HS) Octyl FF − + + Butyl FF − + + Ion Exchange QFF + − + SP FF + − − Mixed Mode HA Type I  + − − HA Type II + − − Abx +− − Hydrophobic Charge- MEP − + − Induction Immobilized Metal-ionChelating + − − Affinity FF

This experiment clearly showed that the desired separation of thebotulinum toxin from other substances present was best achieved by useof hydrophobic type column chromatography. Thus, we found that thebotulinum toxin bound to hydrophobic columns, but that it did not bindto an ion exchange column, such as the Q Sepharose FF column.

Among the hydrophobic columns evaluated, the weakly hydrophobic ButylSepharose FF gave the best resolution. Therefore, either Butyl SepharoseFF in binding mode or Q Sepharose FF in flowthru mode provided apreferred botulinum toxin capturing step.

Thus, we determined that an efficient capturing step can be carried outusing a hydrophobic column, such as the Butyl Sepharose columnchromatography. Presumably, the toxin binds to the Butyl column via ahydrophobic interaction. Prior to this experiment it was unknown that abotulinum toxin complex could be purified toxin directly from clarifiedculture using a hydrophobic chromatography column. We found that theButyl Sepharose Fast Flow column has high binding capacity, allows fastflow rate with low back pressure and is therefore suitable for thecapturing step that requires fast removal of impurities.

Example 10 Four Column APF Chromatographic System and Process forPurifying a Botulinum Toxin Complex

Intermediate and Polishing Purification Steps

Additional (intermediate and polishing) toxin purification steps werecarried out using the toxin-containing fractions obtained from thepreferred Q and Butyl columns of Example 9.

Three types of chromatography columns were found effective for suchfurther purification of the botulinum toxin complex A Hydroxyapatite(HA) type I column was a preferred column we used because it showedseparation, but some toxin was found in the flowthru. Gel filtrationwith a Superdex 200 column was a more preferred column to use because itpermitted purification of the 900 kDa botulinum toxin complex from theimpurities, but a minor impurity band was still present on SDS-PAGE.

A most preferred column was a SP Sepharose HP column which we found toseparate the botulinum toxin from impurities with very good resolution.The botulinum toxin was pure after SP Sepharose HP chromatography, basedon analysis by SDS-PAGE.

TABLE 4 Summary of Column Chromatography Purification Steps Separationtechnique Resin Summary Mixed mode Hydroxyapatite Toxin in flowthrumode, type I separated some impurities. Gel filtration Superdex 200Partially purified toxin, difficult to scale-up, low productivity. Ionexchange SP Sepharose HP High resolution separation, pure toxinobtained.

Based on the results of Examples 8 and 9, and as shown by Table 4, thefollowing four column chromatography purification process was developed.

1. use of a Q Sepharose FF column for initial purification of aclarified culture. In this step impurities bound to the column and thetoxin flowed through the column;2. the eluent from the Q Sepharose FF column step 1 was then passedthrough a Butyl Sepharose FF column. The toxin bound to the column andwas eluted off with a suitable buffer;3. the eluent from the Butyl Sepharose FF was then passed through aHydroxyapatite type I column. Impurities bound to the column and thetoxin flowed through the column;4. the eluent from the Hydroxyapatite type I was then passed through anSP Sepharose column. The toxin bound to the column and was eluted offwith a suitable buffer.

This four column toxin purification process can be summarized as:

APF clarified culture=>Q(flowthru)=>Butyl(binding)=>HA (flowthru)=>SP (binding)=>purified toxin complex

This four column bulk botulinum toxin complex process allowed directloading of filtered culture supernatant onto the Q column (step 1). Theflowthru was supplemented with ammonium sulfate to 0.8M before thesecond step of loading onto the Butyl column. For the third step, thebutyl eluate was loaded onto the HA column directly, while the flowthruof the HA was diluted 4 times with deionized water and the pH wasadjusted to 4.0 before loading onto the SP column for the fourth columnstep. This four column process required minimal sample handling at eachstep, and ensured that the toxin was exposed to mild bufferingconditions throughout the four steps of this purification process.

A scale up of the four column purification process set forth above wasused carried out upon 680 ml of filtered culture supernatant obtainedfrom an APF botulinum toxin type A fermentation process. The results(see Table 5) show that this four column process resulted in highly in ahigh yield of highly purified botulinum toxin type A complex.

TABLE 5 Results of a Scale Up Purification using the Four ColumnPurification process. Toxin yield ~30 mg per L culture based on UV andHc- ELISA. Toxin purity >98%, monodisperse, 900 kDa complex basal onSEC-HPLC and LS. Pure on SDS PAGE, western blotting conforms tostandard. Toxin 3-5 × 10⁷ MLD₅₀ units per mg based on potency mousetoxicity, assays.

Example 11 Additional Multi-Column APF Chromatography Processes forPurifying a Botulinum Toxin Complex

Using the same procedures set forth in Examples 9 and 10 additionalcolumn combinations were evaluated. It was determined that each of thefollowing four additional column combinations provided APF methods forobtaining highly purified botulinum toxin complex, as determined bySDS-PAGE.

1. Q (flowthru)=>Butyl=>SP2. Butyl=>Q or HA (flowthru)=>SP3. Butyl=>SP=>Q or HA (flowthru)

4. Butyl=>SP

The purified toxins were further analyzed by SEC-HPLC with lightscattering, capillary electrophoresis, residual DNA assay, H-ELISA, andMLD50. No significant differences were found among the toxins from thefour different processes set forth above. The results are summarized inTable 6.

TABLE 6 Quality summary of toxin samples purified by different APFprocesses 1.4. above. SEC-HPLC/LS Purity >99%, purer than BCC2030, butless homogeneous than BCC2030. Capillary Identical to one another,similar to 19P and eletrophoresis 20P Research Grade APF Toxin, butslightly different from BCC2030. Picogreen DNA assay 2-6 ng/ml,significantly lower than BCC2030. Mouse toxicity assay, Toxin potency3.1-4.8 × 10⁷ MLD₅₀ units/mg Hc-ELISA toxin (by UV), or 3.8-12 × 10⁷MLD₅₀ units toxin (by Hc-ELISA). Silver staining SDS- Identical to oneanother. PAGE

Example 12 Two Column APF Chromatography Process for Purifying aBotulinum Toxin Complex

Based on the results obtained in Example 11 a two column (Butyl=>SP)column chromatography process was selected for further development.

Optimization of the First Step: Butyl Sepharose FF Toxin Capture

Feed: Feed is to the clarified culture loaded on the column. Sinceammonium sulfate can affect the buffer pH, the use of NaCl to replaceammonium sulfate in Butyl column was evaluated. We found that additionof NaCl to the feed sufficient to 2M NaCl allowed the botulinum toxincomplex to bind to the butyl column. Subsequently, we determined thatfeed at a 4M NaCl increased the binding of botulinum toxin complex tothe Butyl column, such that the yield of toxin from the Butyl column wasincreased by 30% to 50%, as determined by Hc-ELISA, as compared to useof feed at 2M NaCl.

The addition of NaCl to the clarified culture (the feed) caused a smallpH shift. However, the acceptable feed pH was established between pH 5and pH 6 and the final pH of the feed after NaCl addition was within pH5 and pH 6. Hence the preferred feed to use in this first step of a twocolumn purification process has a 4M NaCl concentration and is at pH5-6. Solid NaCl was added to the clarified culture directly to obtainthe 4M NaCl concentration and this feed was then added to the Butylcolumn. The bound toxin was eluted from the column using a 1M NaClelution buffer.

It was surprising that most of impurity proteins could be washed awayfrom the column and most of toxin bound to the column could be elutedwith a 1M NaCl buffer because column purification processes typicallyconsist of 3 or more columns: except for an affinity column process. Wedetermined that this butyl column is unique as it has the ability toremove many of the impurity proteins. Thus, after use of this column thebotulinum toxin complex purity was approximately 50%.

A wash step was then carried out to remove impurities from a column. Theimpurities in the column came from the clarified culture feed(containing 4M NaCl) used. The optimized washing steps were: 1) Wash #1:5 CV of 50 mM NaPi, 4M NaCl, pH 6.0, and 2) Wash #2: 12 CV of 50 mMNaPi, 2M NaCl, pH 6.0. When 12 CV and 5 CV were compared, it was foundthat 5 CV is not sufficient in removing the impurities, while the washis to remove impurities after loading the clarified culture in thiscase.

Elution (to remove toxin bound to a column). Toxin elution with 1.2M,1.0M and 0.8M NaCl were evaluated. It was chosen to elute toxin with 1MNaCl in 50 mM NaPi, pH 6.0, based on toxin recovery and impurityremoval.

Low salt wash: After elution, the column was further washed with 50 mMNaPi, pH 6.0 to remove residual impurities bound to the column for thecharacterization of purification process.

Cleaning: the column was cleaned with 3 CV of 0.1N NaOH to inactivateany residual toxin before the disposal of used resin.

Running flow rate: The typical flow rate was 100 cm/h. The loading flowrate was between 90 cm/h and 120 cm/h depending on the back pressure.

Loading capacity: Typical loading capacity was 12.7 ml culture per mlbed, or at production scale, 10 L culture for 785 ml resin bed (BPG 100column at 10 cm bed height).

Bed height: All columns were packed with standard 10 cm bed height.

Optimization of the Second Step: SP Sepharose HP Purification

Feed conditioning: The Butyl eluate was diluted 5 times with 20 mM Nacitrate buffer, pH 4.0, and the feed pH was adjusted to 4.0. The fivetimes dilution step was carried out to condition the hydrophobicinteraction chromatography eluent for use in ion exchangechromatography. We found that the optimal feed pH for best toxinrecovery was within the range of pH 4.0±0.2.

Wash step: After loading, the column was washed with 1) 5 CV of 20 mM Nacitrate, pH 4.0, followed by 2) 3-5 CV of 20 mM Na citrate, 300 mM NaCl,pH 4.0 to remove impurities before the elution of bound toxin.

Elution step: The toxin was eluted with 20 mM Na citrate 400 mM NaCl, pH4.0.

High salt washing step: After elution, the column was further washedwith 20 mM Na citrate, 1M NaCl, pH 4.0 to remove strongly boundimpurities.

Column cleaning: The column was cleaned with ˜3 CV of 0.1N NaOH toinactivate residual toxin before the disposal of used resin.

Flow rate: The typical flow rate was 100 cm/h.

Load: The entire Butyl eluate was loaded onto the SP column.

Bed height: All columns were packed with standard 10 cm bed height.

Detailed operating procedures carried out with regard to this two columnbotulinum toxin complex purification process set forth in this Example12 are set forth below.

1. Butyl Hydrophobic Interaction Column Materials and Reagents Used

Chromatography System: AKTA purifier 100, Amersham Biosciences

Resin Type: Butyl Sepharose FF, Amersham Pharmacia Detection: UV (280nm) Equilibration Buffer/Wash Buffer #1: 50 mM NaPi, 4 M NaCl, pH 6.0Wash Buffer #2: 50 mM NaPi, 2 M NaCl, pH 6.0 Elution Buffer: 50 mM NaPi,1 M NaCl, pH 6.0 Low Salt Wash Buffer: 50 mM NaPi, pH 6.0 CleaningSolution: 0.1 N NaOH Titration Buffer: 500 mM NaPi, pH 7.2 ProcedureColumn Packing and Conditioning

Equilibrate the column with at least 5-10 CV of Equilibration Buffer oruntil outlet pH is equivalent to inlet pH.

Sample Preparation

Measure the pH of the starting material.Add solid NaCl to the clarified culture to the final NaCl concentrationto 4 M. Addition of 4M NaCl is an example of how to condition theclarified culture for use of the clarified culture as a feed liquid inhydrophobic interaction chromatography. Adjust the pH to 5.0 to 6.0 ifneeded with Titration Buffer.

Column Loading

Load the clarified culture (containing 4M NaCl) and collect the flowthrough fraction for analysis.

Column Wash #1 (4 M NaCl Wash)

Wash the column proteins with 5 CV of Equilibration Buffer to removeimpurity. Collect the wash fraction for analysis and record the volume.

3.5. Column Wash #2 (2 M NaCl Wash)

Wash the column with 15 CV of Wash Buffer #2 to remove additionalimpurity proteins. Collect the wash fraction for analysis and record thevolume.

Elution (1 M NaCl Toxin Peak Elution)

Elute the bound toxin with 5 CV of Elution Buffer. Monitor the 280 nmabsorbance of eluate, begin the collection of eluate when the 280 nmabsorbance starts to increase and stop the collection of the eluate peakwhen the 280 nm absorbance reaches the baseline. Record the volume oftoxin elution fraction.

Low Salt Wash (0 M NaCl Impurity Peak Elution)

Wash the column with 4 CV of Low Salt Wash Buffer to remove residualimpurity proteins. Collect the fraction for analysis and record thevolume.

Column Cleaning (0.1 N NaOH)

Clean the column with 3 CV of Cleaning Buffer to inactivate the residualtoxin before the disposal of used resin.

2. SP Cation Exchange (Post Butyl) Column Materials and Reagents Used

Chromatography System: AKTA purifier 100, Amersham Biosciences

Resin Type; SP Sepharose HP, Amersham Pharmacia Detection: UV (280 nm)Dilution, Equilibration and Wash Buffer #1: 20 mM NaCitrate, pH 4.0 WashBuffer #2: 20 mM NaCitrate, 300 mM NaCl, pH 4.0 Elution Buffer: 20 mMNaCitrate, 400 mM NaCl, pH 4.0 High Salt Buffer: 20 mM NaCitrate, 1 MNaCl, pH 4.0 Cleaning Solution. 0.1 N NaOH Procedure Column Packing andConditioning

Equilibrate the column with 5-10 CV of Equilibration Buffer or untiloutlet pH is equivalent to inlet pH.

Sample Preparation

Dilute one volume of 1M NaCl Butyl eluate with 4 volume of DilutionBuffer. Measure the conductivity and pH of the load. Adjust the pH to4.0 if needed.

Column Loading

Apply the above diluted Butyl eluate to SP column and collect the flowthrough fraction.

Column Wash #1 (Equilibration Buffer Wash)

Wash the SP column with 5 CV of Equilibration Buffer. Continue tocollect the eluate as flow through fraction.

Column Wash #2 (300 mM NaCl Wash)

Wash the SP column with 4 CV of Wash Buffer #2 to remove impurityproteins. Record the volume of the wash #2 fraction.Elution (400 mM NaCl elution) Elute the bound toxin with 3 CV of ElutionBuffer. Monitor the 280 nm absorbance of eluate, begin the collection ofeluate when the 280 nm absorbance starts to increase and stop thecollection of the eluate peak when the 280 nm absorbance reaches thebaseline. Record the volume of toxin elution fraction.

High Salt Elution (1 M NaCl)

Elute the strongly bound impurity proteins with 3 CV of High SaltBuffer. Collect the fraction for analysis and record the volume

Column Cleaning

Clean the SP column with 3 CV of Cleaning Solution to inactivate theresidual toxin before the disposal of used resin.

Example 13 Robustness of the Two Column APF Chromatography Process forPurifying a Botulinum Toxin Complex

The robustness of the two column method of Example 12 was studied in aseries of experiments, as set forth below.

Culture pH

The effect of culture pH on toxin purification was evaluated. A studyusing cultures grown at pH 5.5 and pH 6.5 as the starting material forthe purification was performed, and it was found that the recovery fromthe pH 6.5 culture was slightly lower than that from the pH 5.5 culture,based on Hc-ELISA results.

Storage Time

Toxin was purified from a culture grown at pH 5.5 on the day ofharvesting and after 4-day storage of the culture at 2-8° C. Nodifference was found, based on toxin recovery, Butyl and SPchromatograms, SDS-PAGE, and Hc-ELISA results.

Column Binding Capacity

The proposed load on the Butyl column was 12.7 ml culture per ml resin,or 10 L culture for BPG100 column (with 10 cm bed height). Butyl and SPcolumns were tested by loading 4× more culture. SDS-PAGE and Hc-ELISAresults indicated little toxin in the flowthru fractions for both Butyland SP columns. The capacity of Butyl and SP column is at least fourtimes greater than that of the current load. The toxin in SP eluate waspure on SDS-PAGE. The recovery of Butyl column is 48% and the recoveryof SP column was 74%, based on Hc-ELISA. The overall yield is 16 mgtoxin per L culture, based on UV result.

Process Hold Time

After harvesting, the culture was processed through Butyl column on thesame day or after overnight storage. Butyl eluate was normally storedovernight before loading onto the SP column. A preliminary study showedthat the Butyl eluate was stable for up to 4 days, which gave identicalchromatogram and SDS-PAGE patterns. The stability of SP eluate wasevaluated by capillary electrophoresis (CE) and SEC-HPLC. The resultsshowed no difference among samples stored for up to 2 days. The recoveryof toxin after filtration was also evaluated for these samples. Toxinrecovery was slightly decreased on day 2 compared to day 0, but it wasnot clear whether such decrease was due to storage or experimentalvariation.

Cell Density of Culture

Two times concentrated culture and 2× diluted culture were evaluated byButyl column chromatography to study the effect of culture cell densityon toxin purification. The chromatograms from both runs lookedidentical. The impurity and toxin profile from both runs were identicalon SDS-PAGE. The Hc-ELISA results (Table 7) showed that the mass balancefrom both runs were >90%, while the recovery of 2× concentrated culturewas significantly lower than that of 2× diluted culture Twenty-ninepercent toxin was lost before toxin elution for 2× concentrated culture,compared with 4% loss for 2× diluted culture.

TABLE 7 APF toxin mass balance analyzed by Hc-ELISA. Mass 2M 1M 0M Runbalance FT Wash Elution Elution 2x cement 91% 11% 18% 53%  9% 2x diluted97%  0%  4% 74% 19%

Bioburden Studies

Bioburden was monitored at different steps of the process. Samples ofButyl load, Butyl eluate after 3 day storage. SP load, SP eluate, and SPeluate after overnight storage were evaluated. Some contaminants werenoted (˜<1 CFU/ml to 35 CFU/ml). The sample with the highest number ofcontaminants was the Butyl eluate. Contaminants may be due to theuncontrolled environment in which purification process was performed.

Effect of 4M NaCl

In order to evaluate the effect of 4M NaCl on the toxin in culture, theculture containing 4M NaCl was kept at 4° C. overnight and then Butyland SP columns were performed. The chromatographic result, SDS-PAGE andHc ELISA showed there was no effect of 4M NaCl on the toxin in theculture after overnight storage.

Culture media (311 vs 5:1:1)

Two point five liters of 5:1:1 and 3:1:1 cultures were processed. Thetoxin recovery for each of the purifications analyzed by Hc-ELISA issummarized in Table 8. The toxin purified from both cultures was pure onSDS-PAGE, which indicates that the process developed with 3:1:1 culturecan be used to purify toxin from 5:1:1 culture.

TABLE 8 Toxin recovery based on Hc-ELISA Step 3:1:1 culture 5:1:1culture Butyl 46% 43% SP 63% 44%

Working pH for SP Sepharose HP Chromatography

SP Sepharose HP chromatography was carried out at different pH values:3.5, 4.2, and 4.5. It was found that pH 3.5 caused toxin precipitationin the column, no toxin was eluted with 400 mM NaCl and very littletoxin came out with 1M NaCl. At pH 4.5, toxin did not bind to the SPcolumn. Preliminary results obtained at pH 4.2 showed that the toxin didnot bind as strongly as at pH 4.0 and was eluted as a broad peak afterthe wash peak at 300 mM NaCl. The results indicate that the pH at thisstep was critical and that the optimal pH range was narrow.

Example 14 Evaluation of Two Column APF Chromatography Process forPurifying a Botulinum Toxin Complex

Various eluents from each of the two columns of the purification processof Example 12 were evaluated as set forth below.

A. Butyl Sepharose FF chromatography

Filtered 3:1:1 culture was used as the feed for this experiment. Beforeloading the feed (clarified culture obtained from a Schantz fermentationof a Clostridium botulinum type A [Hall strain]) onto the ButylSepharose FF column (XK50/10, column diameter 5 cm, bed height 10 cm,column volume: 196 ml), 584.4 g of NaCl was added to 2500 ml of culturewith stirring for 30 min. Atypically, the feed pH was adjusted to 5.81and the running flow rate was set at 92 cm/h (normal flow rate is 100cm/h). The loading volume was 2800 ml.

After loading, the column was washed with 5 CV or 1000 ml of 50 mM NaPi,4M NaCl, pH 6.0, followed by 15 CV or 3000 ml of 50 mM NaPi, 2M NaCl, pH6.0. The bound botulinum toxin type A complex was then eluted from thecolumn with 5 CV or 1000 ml of 50 mM NaPi, 1M NaCl, pH 6.0. After theelution of the botulinum toxin complex, the strongly bound impuritieswere washed off the column with 4 CV or 800 ml of 50 mM NaPi, pH 8.0.The column was next washed with 2 CV (400 ml) of 0.1N NaOH to inactivateresidual toxin before the disposal of used resin. The chromatogram ofthe toxin eluent is shown in FIG. 3.

FIG. 3 shows that the Butyl column used can provide good separation ofbotulinum toxin complex from impurities present with it in the clarifiedculture feed liquid. As measured by UV280 nm, FIG. 3 shows the flowthrough peak and the peaks of 2M NaCl, 1M NaCl, 0M NaCl and 0.1N NaOH.Based on the peak size, it was determined that most impurities wereremoved in the flow through fraction. A significant amount of impuritieswere also removed in 2M NaCl fraction before the elution of toxin in the1M NaCl fraction.

FIG. 3 is a chromatograph obtained from passage of an APF clarifiedculture (a 3.1.1 culture) through a Butyl hydrophobic interactioncolumn. The X axis represents the volume in ml of liquid (effluent)which has passed through the column. The Y axis represents the UVabsorbance at 280 nm in mAU. In addition, the conductivity (separategraph line) was monitored during chromatography.

As shown by FIG. 3, many protein impurities passed through the column inabout the first approximately 3000 mls. The 4M NaCl and 2M NaCl washesbuffer cause subsequent, though smaller peaks, showing removal ofadditional impurities. Use of the 1M NaCl (at about the 7000 ml volume)caused elution of bound toxin complex from the column and this was thefraction loaded onto the second column.

B. SP Sepharose HP Chromatography

The axes in FIG. 4 are the same as they are for FIG. 3. The stepscarried out to obtain the FIG. 4 chromatograph were as follows:

(1) one hundred ml of the Butyl eluate obtained from Example 12 (theButyl column eluent resulting from FIG. 3) was diluted with 400 ml of 20mM Na citrate buffer at pH 4.0 (a five times dilution therefore). The pHof this diluted Butyl eluent was 4.1. (2) four hundred and sixty-six mlof this feed was then loaded onto the SP Sepharose HP column (XK26/10,column diameter 2.6 cm, bed height 10 cm, column volume: 53 ml).(3) after loading the column was washed (at about the volume 450 mlpoint on the x axis of FIG. 4) with 5 CV or 250 ml of 20 mM Na citrate,pH 4.0.(4) the column was then washed with 4 CV or 200 ml of 20 mM Na citrate,300 mM NaCl, pH 4.0 (at about the volume 725 ml point on the x axis ofFIG. 4) n.(5) the column bound botulinum toxin complex toxin was then eluted with3 CV or 150 ml of 20 mM Na citrate, 400 mM NaCl, pH 4.0 (at about thevolume 925 ml point on the x axis of FIG. 4).(6) after elution of the column bound toxin complex, the column wasfurther washed with 3 CV or 150 ml of 20 mM Na citrate, 1M NaCl, pH 4.0to elute strongly bound impurities (at about the volume 1050 ml point onthe x axis of FIG. 4).(7) the column was then cleaned with 3 CV or 150 ml of 0.1N NaOH (justafter the volume 1200 ml point on the x axis of FIG. 4).

The FIG. 4 chromatogram shows elution of a botulinum toxin type Acomplex (about 900 kDa molecular weight) just before the 1000 ml volumepoint on the x axis of FIG. 4.

FIG. 4 shows that high purified botulinum toxin complex can be obtainedby use of the SP sepharose column subsequent to the Butyl column. FIG. 3shows that there was a broad flow through peak, a small 300 mM NaCl washpeak, 400 mM toxin elution peak and 1M NaCl cleaning peak. As analyzedby SDS-PAGE in FIG. 58, there was no visible protein band in flowthrough fraction, some impurity protein bands in 300 mM NaCl washfraction and 1M NaCl cleaning fraction. Toxin was eluted in 400 mM NaClelution fraction.

C. Analytical Results:

SDS-PAGE: The elution fractions from the Butyl and SP columnchromatography columns were analyzed by SDS-PAGE and the typical resultis shown in FIG. 5A (Butyl column) and FIG. 4B (SP column).

FIGS. 5 and 6 are gel electrophoresis records obtained by use of reducedSDS-PAGE. The left had side of the FIGS. 5 and 6 gel electrophoresisrecords is marked vertically with ascending molecular weights inthousands of Daltons (kDa). The numbers 1 to 8, 1 to 7 or 1 to 8 isFIGS. 5 and 6 represent the fractions loaded onto the gels.

In FIG. 5A: item 1 (gel lane 1) “Mark 12” is the Novex molecular weightmarker of standard molecular masses; lane 2 is the clarified culturefeed liquid; lane 3 is an aliquot from the wash resulting from use ofthe flow through (“FT”) and 4M wash in the Butyl column; lane 4 is analiquot from use of the 2M wash; lane 5 is an aliquot from the tailfraction of the 2M wash; lane 6 is an aliquot from the fraction of 1Melution; lane 7 is an aliquot from the tail fraction of 1M elution, and;lane 8 is an aliquot from the 0M wash.

FIG. 5A shows that the Butyl column removed many impurities (see columns3-5 in FIG. 5A) and provided initially purified botulinum toxin (seecolumns 6-8 in FIG. 5A).

In FIG. 5B: item 1 (gel column 1) “Mark 12” is the same molecular weightmarker used in FIG. 5A; column 2 is the diluted Butyl column eluent:column 3 is an aliquot of the column flow through; column 4 is analiquot from the 300 mM wash; column 5 is an aliquot from eluant fromthe column; column 6 is an aliquot from the 1M wash. FIG. 5B shows thatuse of an SP column subsequent to use of a Butyl column provided highlypurified botulinum toxin (see column 5 in FIG. 5B).

Hc-ELISA

Toxin concentration was analyzed by Hc-ELISA, an ELISA assay todetermine the toxin concentration based on the concentration of toxinheavy chain, and toxin mass balance during the purification wasestimated Table 9 shows the toxin concentration and step recovery duringButyl and SP column steps from a typical purification run. The overallrecovery after Butyl and SP was 28.6%.

SEC-HPLC

The results from SEC-HPLC showed that the step recovery for SPchromatography was 42.9%, compared to 62.5% from Hc-ELISA. This showsthat the recovery of botulinum toxin after the SP column step wasapproximately 50%.

Normalized Yield

The toxin yield was normalized as 22.3 mg (by SEC-HPLC) or 23.4 mg (byHc-ELISA) per L culture after Butyl chromatography, and 9.6 mg (bySEC-HPLC) or 8.9 mg (by Hc-ELISA) per L culture after SP chromatographyfrom one run. Thus, using our two column system and process set forthherein, between about 50 mg to about 90 mg of botulinum toxin complexcan be purified from each 10 L of fermentation medium clarified culture(as obtained for example from the Example 6 or Example 72 fermentationprocesses).

TABLE 9 Toxin concentration and mass balance in typical Butyl and SPchromatography steps. Toxin Volume Conc. Amt. % (ml) (μg/ml) (mg)Recovery Butyl samples Butyl Load 2800 45.5 127.4 100 Flowthru and Wash2634 N/A N/A N/A 2M NaCl Wash Peak 336 32.5 10.9 8.6 1M NaCl ElutionPeak 404 144.5 58.4 45.8 1M NaCl Post Elution 443 N/A N/A N/A 0M NaClWash 369 19 7.0 5.5 SP Samples SP Load 466 19 8.9 100.0 Flowthru 708 N/AN/A N/A 300 mM Wash Peak 54 N/A N/A N/A Elution Peak 35 158 5.5 62.5 1MWash Peak 13 N/A N/A N/A Cleaning Peak 44 N/A N/A N/A

Example 15 Process for Post Column Chromatography Toxin ComplexStabilization and Storage

1. Development Rationale

After column chromatography, it is preferred to transfer the purifiedbotulinum toxin complex into a stable buffer at a desired concentrationby a UF/DF step, followed by sterile filtration to thereby obtain atoxin suitable for use in a compounding of a botulinum toxinpharmaceutical composition. The purified botulinum was stored either ina soluble form in acetate buffer or as an ammonium sulfate suspension.

2. UF/DF Step

A polyethersulfone Biomax-10 membrane (NMWCO: 10 kDa, Millipore) wasused in the UF/DF step. 50 mM NaAc, pH 4.0 was chosen as thediafiltration buffer. The SP eluate was ultrafiltered to ˜1 mg/mi, thendiafiltered with 8 diafiltration volumes (DV) of 50 mM NaAc, pH 4.0.

Ultrafiltration (UF) is a process for separating extremely smallparticles and dissolved molecules from fluids. The primary basis for theseparation is molecular size although secondary factors such as moleculeshape and charge can play a role. Materials ranging in size from 1,000to 1,000,000 molecular weight are retained by ultrafilter membranes,while salts and water pass through. Colloidal and particulate matter canalso be retained.

Diafiltration (DF) is the fractionation process that washes smallermolecules through a membrane and leaves larger molecules in theretentate without ultimately changing concentration. F can be used toremove salts or exchange buffers. OF can also remove ethanol or othersmall solvents or additives There are several ways to performdiafiltration. In continuous diafiltration, the diafiltration solution(water or buffer) is added to the sample feed reservoir at the same rateas filtrate is generated. In this way the volume in the sample reservoirremains constant, but the small molecules (e.g. salts) that can freelypermeate through the membrane are washed away. Using salt removal as anexample, each additional diafiltration volume (DV) reduces the saltconcentration further. (A diafiltration volume is the volume of samplebefore the diafiltration solution is added.) Using 5 diafiltrationvolumes will reduce the ionic strength by ˜99% with continuousdiafiltration. In discontinuous diafiltration, the solution is firstdiluted and then concentrated back to the starting volume. This processis then repeated until the required concentration of small molecules(e.g. salts) remaining in the reservoir is reached. Each additionaldiafiltration volume (DV) reduces the salt concentration further. Adiafiltration volume is the volume of sample before the dilutingsolution is added. Using 5 diafiltration volumes will reduce the ionicstrength by ˜96% with discontinuous diafiltration. Continuousdiafiltration requires less filtrate volume to achieve the same degreeof salt reduction as discontinuous diafiltration.

3. 0.22 μm Filtration Step

The low-protein-binding 0.22 μm cellulose acetate (CA) vacuum bottle-topfilter was selected for the filtration step

4. Ammonium Sulfate Precipitation Step

Ammonium sulfate precipitation was then carried out: 3.5M ammoniumsulfate was added to the 0.22 μm filtered toxin solution with gentlestirring until the first appearance of opalescence. The purified bulktoxin was then stored at 2-8° C.

5. Results from a Typical Post-Column Process

SP eluate was concentrated from 70.5 ml to 18 ml using PelliconBiomax-10 (50 cm² surface area, Millipore) on a Labscale TFF system(Millipore) and diafiltered with 8DV of 50 mM NaAc, pH 4.0. Theretentate (post-UF/DF fraction) was collected and was filtered withCorning 0.22 μm CA filter (Corning 431154). The UF/DF system was rinsedwith acetate buffer. The rinse fraction was collected. Ten ml of thepost 0.22 μm filtrate was stored at 2-8° C. for stability studies. Eightml of the post 0.22 μm filtrate was subjected to ammonium sulfateprecipitation A total of 2.8 ml of 3.5 M ammonium sulfate was added intothe filtrate until it became opalescent.

Toxin recovery was estimated based on UV measurement, which is shown inTable 10. SDS-PAGE results are shown in FIG. 4.

In FIG. 6 the lanes shown represent:

Lane 1 is M12, molecular weight standardsLane 2 is SP column eluateLane 3 is UF/DF retentate: UF/DF retentate after UF/DF of SP eluatediluted to the same amount of loaded protein as Lane 2, for comparisonLane 4 is UF/DF rinse solution from rinsing UF/DF membrane aftercompletion of UF/DF membraneLane 5 is post 0.2 μm filtration; after UF/DF process and after thesample was filtered with the 0.22 μm filterLane 6 is post column ammonium sulphate suspension: after 0.22 μm filterfiltration, the sample was precipitated with ammonium sulphate becausethe botulinum toxin complex is stable in ammonium sulphateLane 7 is UF/DF retentate (same as lane 3), but undiluted, to show thedetails

FIG. 6 tells us that the post column purification process steps ofUF/DF, 0.22 μm filtration, and ammonium sulphate precipitation do notaffect the purity of the botulinum toxin complex, as determined bySDS-PAGE analysis. Significantly, the MLD₅₀ results showed that thepotency of the purified bulk botulinum toxin complex was 2.9-37×10⁷MLD₅₀ units/mg.

TABLE 10 Toxin recovery based on UV measurement Toxin conc. Total by UVVol. toxin Recovery Fraction (mg/ml) (ml) (mg) (%) SP eluate 0.389 70.527.4 100 (defined) Post UF/DF 1.260 18.0 22.7 82.8 UF/DF rinse 0.22014.0 3.1 11.3 Post filtration 1.270 18.0 22.8 83.2 Post AS ppt* N/A(~0.94) ~10.8 N/A N/A *from 8 ml post filtration fraction.

FIG. 7 is a flowchart of a preferred animal protein free, two columnchomatographic method for purifying a botulinum toxin type A complex.This is a robust, scalable and cGMP compliant process for obtainingpurified Clostridium botulinum toxin 900 kDa complex. In FIG. 7 it canbe noted that the Butyl eluate is conditioned for ion exchangechromatography by a five times dilution with a pH 4 sodium citratebuffer.

The FIG. 7 process can also be used to obtain pure (i.e. 150 kDabotulinum toxin free of the non-toxin complex proteins) by loading theSP column eluent onto an ion exchange column in a pH 8 buffer todisassociate the non toxin complex proteins from the 150 kDa botulinumtoxin molecule, thereby providing (in the flow through from the column)a botulinum toxin type A (neurotoxic component) with an approximately150 kD molecular weight, and a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater. This process can also be used to obtain other non toxincomponents of botulinum toxin complex (i.e. non toxin hemagluttininproteins and/or non toxin non hemaglutinin proteins) by dissociating thecomplex into its components and next purifying the dissociatedcomponents

The purified toxin complex obtained by our process meets or exceeds thespecifications set forth in Table 1. Additionally, the typical yield wasapproximately 100 mg of 900 kDa toxin complex from a 10 L cell culture,which is higher than the yield obtained from a Schantz (non-APF)process.

Advantages of our invention include:

1. No component or substance derived from animal source is used in theprocess. Specifically, use of DNase and RNase are eliminated.2. More than about 50 mg per purified botulinum toxin type A complexwith the characteristics set forth in Table 1 can be obtained per 10liters of fermentation medium.3. The purified toxin is obtained from a process which is robust,scalable, validatable, and cGMP compliant. Robust means the process isreproducibility even upon an about ±10% change in one or more of theprocess parameters. Validatable means the process consistently yieldpurified toxin with the table 1 characteristics. cGMP means that theprocess can be easily converted to a manufacturing process that complieswith FDA required current Good Manufacturing Practices.4. The potency of the final purified botulinum toxin complex meets orexceeds the potency (as determined by the MLD50 assay) of purifiedbotulinum toxin complex obtained from a Schantz or modified Schantzprocess.5. elimination of any precipitation steps to purify a botulinum toxincomplex.

Various publications, patents and/or references have been cited herein,the contents of which, in their entireties, are incorporated herein byreference.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of animal product free systems and processes(including chromatographic botulinum toxin purification processes) arewithin the scope of the present invention.

Accordingly, the spirit and scope of the following claims should not belimited to the descriptions of the preferred embodiments set forthabove.

1-6. (canceled)
 7. A composition comprising: (i) a biologically active,approximately 900 kDa Clostridium botulinum toxin serotype A (BoNT/A)complex; (ii) a buffer; (iii) a salt; and (iv) a hydrophobic interactionchromatography (HIC) media, wherein: the composition has a pH in a rangeof about 5.0 to about 6.8; the concentration of the salt is sufficientto bind the biologically active, approximately 900 kDa BoNT/A complex tothe HIC media; and the biologically active, approximately 900 kDa BoNT/Acomplex is bound to the HIC media.
 8. The composition of claim 7,wherein the composition has a pH in a range of about 5.5 to about 6.8.9. The composition of claim 7, wherein the biologically active,approximately 900 kDa BoNT/A complex is obtained from an animalprotein-free (APF) fermentation process.
 10. The composition of claim 7,wherein the salt comprises sodium chloride.
 11. The composition of claim7, wherein the salt comprises ammonium sulfate.
 12. The composition ofclaim 7, wherein the HIC media comprises phenyl sepharose media.
 13. Thecomposition of claim 7, wherein the HIC media comprises butyl sepharosemedia.
 14. The composition of claim 7, wherein the HIC media comprisesoctyl sepharose media.
 15. A composition comprising: (i) a biologicallyactive, approximately 900 kDa Clostridium botulinum toxin serotype A(BoNT/A) complex; (ii) a buffer; (iii) a salt; and (iv) a hydrophobicinteraction chromatography (HIC) media, wherein: the composition has apH in a range of about 5.0 to about 6.8; the concentration of the saltis sufficient to bind the biologically active, approximately 900 kDaBoNT/A complex to the HIC media; the biologically active, approximately900 kDa BoNT/A complex is bound to the HIC media; and the composition isessentially animal protein free (APF).
 16. The composition of claim 15,wherein the composition has a pH in a range of about 5.5 to about 6.8.17. The composition of claim 15, wherein the salt comprises sodiumchloride.
 18. The composition of claim 15, wherein the salt comprisesammonium sulfate.
 19. The composition of claim 15, wherein the HIC mediacomprises phenyl sepharose media.
 20. The composition of claim 15,wherein the HIC media comprises butyl sepharose media.
 21. Thecomposition of claim 15, wherein the HIC media comprises octyl sepharosemedia.
 22. A composition comprising: (i) a biologically active,approximately 900 kDa Clostridium botulinum toxin serotype A (BoNT/A)complex; (ii) a buffer; (iii) a salt; and (iv) a hydrophobic interactionchromatography (HIC) media, wherein: the composition has a pH in a rangeof about 5.0 to about 6.8; the concentration of the salt is sufficientto bind the biologically active, approximately 900 kDa BoNT/A complex tothe HIC media; the biologically active, approximately 900 kDa BoNT/Acomplex is bound to the HIC media; and the HIC media comprises phenylsepharose media.
 23. The composition of claim 22, wherein thecomposition has a pH in a range of about 5.5 to about 6.8.
 24. Thecomposition of claim 22, wherein the biologically active, approximately900 kDa BoNT/A complex is obtained from an animal protein-free (APF)fermentation process.
 25. The composition of claim 22, wherein thecomposition is essentially animal protein free (APF).
 26. Thecomposition of claim 22, wherein the salt comprises sodium chloride. 27.The composition of claim 22, wherein the salt comprises ammoniumsulfate.
 28. A composition comprising: (i) a biologically active,approximately 900 kDa Clostridium botulinum toxin serotype A (BoNT/A)complex; (ii) a buffer; (iii) a salt; and (iv) a hydrophobic interactionchromatography (HIC) media, wherein: the composition has a pH in a rangeof about 5.0 to about 6.8; the concentration of the salt is sufficientto bind the biologically active, approximately 900 kDa BoNT/A complex tothe HIC media; the biologically active, approximately 900 kDa BoNT/Acomplex is bound to the HIC media; and the HIC media comprises butylsepharose media.
 29. The composition of claim 28, wherein thecomposition has a pH in a range of about 5.5 to about 6.8.
 30. Thecomposition of claim 28, wherein the biologically active, approximately900 kDa BoNT/A complex is obtained from an animal protein-free (APF)fermentation process.
 31. The composition of claim 28, wherein thecomposition is essentially animal protein free (APF).
 32. Thecomposition of claim 28, wherein the salt comprises sodium chloride. 33.The composition of claim 28, wherein the salt comprises ammoniumsulfate.